Human Nkx-2.2 polypeptide-encoding nucleotide sequences

ABSTRACT

The present invention features a human Nkx-2.2 polypeptide and nucleotide sequences encoding Nkx-2.2 polypeptides. In a particular aspect, the polynucleotide is the nucleotide sequence of SEQ ID NO:1. In addition, the invention features polynucleotide sequences that hybridize under stringent conditions to SEQ ID NO:1. In related aspects the invention features expression vectors and host cells comprising polynucleotides that encode a human Nkx-2.2 polypeptide. The present invention also relates to antibodies that bind specifically to a human Nkx-2.2 polypeptide, and methods for producing human Nkx-2.2 polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.08/900,510, filed Jul. 25, 1997, which application is incorporatedherein by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made, at least in part, with the grants from theNational Institutes of Health (Grant Nos. DK-41822, DK-20595, DK-44840,and DK-47486). Thus, the U.S. government may have certain rights in thisinvention.

FIELD OF THE INVENTION

The invention relates generally to the field of nucleotide sequencesencoding gene products having a role in pancreatic development, neuraldevelopment, diabetes, depression, and/or obesity.

BACKGROUND OF THE INVENTION

Diabetes mellitus is the third leading cause of death in the U.S. andthe leading cause of blindness, renal failure, and amputation. Diabetesis also a major cause of premature heart attacks and stroke and accountsfor 15% of U.S. health care costs. Approximately 5% of Americans, and asmany as 20% of those over the age of 65, have diabetes.

Diabetes results from the failure of the β-cells in the islets ofLangerhans in the endocrine pancreas to produce adequate insulin to meetmetabolic needs. Diabetes is categorized into two clinical forms: Type 1diabetes (or insulin-dependent diabetes) and Type 2 diabetes (ornon-insulin-dependent diabetes). Type 1 diabetes is caused by the lossof the insulin-producing β-cells. Type 2 diabetes is a more stronglygenetic disease than Type 1 (Zonana & Rimoin, 1976 N. Engl. J. Med.295:603), usually has its onset alter in life, and accounts forapproximately 90% of diabetes in the U.S. Affected individuals usuallyhave both a decrease in the capacity of the pancreas to produce insulinand a defect in the ability to utilize the insulin (insulin resistance).Obesity causes insulin resistance, and approximately 80% of individualswith Type 2 diabetes are clinically obese (greater than 20% above idealbody weight). Unfortunately, about one-half of the people in the U.S.affected by Type 2 diabetes are unaware that they have the disease.Clinical symptoms associated with Type 2 diabetes may not become obviousuntil late in the disease, and the early signs are often misdiagnosed,causing a delay in treatment and increased complications. While the roleof genetics in the etiology of type 2 diabetes is clear, the precisegenes involved are largely unknown.

Depression and obesity can each be associated with a defect in serotoninproduction, serotonin metabolism, or serotonin-mediatedneurotransmission. Serotonin (5-hydroxytryptamine (5HT)) is a biogenicamine that not only functions as a neurotransmitter (Takaki, et al.,1985 J. Neurosciences 5:1769) and as a hormone, (Kravitz, et al., 1980J. Exp. Biol. 89:159), but also as a mitogen (Nemeck, et al., 1986 Proc.Natl. Acad. Sci. USA 83:674). In its functions as a neurotransmitter,serotonin modulates many forms of synaptic transmission and is believedto exert a number of effects on neuronal growth during earlydevelopment. In addition, serotonin is also believed to modulatenumerous sensory, motor, and behavioral processes in the mammaliannervous system (see Jacobs. in Hallucinogens: Neurochemical, Behavior,and Clinical Perspectives (eds. Jacobs) 183-202 (Raven, N.Y., 1984),Sleight et al., in Serotonin Receptor Subtypes: Basic and clinicalaspects. (eds. Peroutka) 211-227 (Wiley-Liss, New York, N.Y., 1991.Wilkinson et al. in Serotonin receptor subtypes: Basic and clinicalaspects (eds. Peroutka) 147-210 (Wily-Liss, New York, N.Y., 1991)). Inthe cortex, transmission at serotoneurgic synapses contributes toaffective and perceptual states; these synapses represent a major siteof action of psychotropic drugs such as LSD, Jacobs in Hallucinogens:Neurochemical, Behavioral, and Clinical Perspectives, Jacobs, Ed. (RavenPress, New York, 1984), pages. 183-202.

The diverse responses elicited by serotonin are mediated through theactivation of a large family of receptor subtypes, Tecott et al. 1993Curr. Opin. Neurobiol 3,310-315. The complexity of this signaling systemand the paucity of selective drugs have made it difficult to understanddevelopment of serotonin-producing cells and to understand the role ofserotonin in depression and obesity.

Attempts to understand depression and other serotonin-related disordershave focused upon understanding the development of the brain.Development of the vertebrate forebrain is an elaborate process thatgives rise to a variety of essential structures including the cerebralcortex, basal ganglia, hypothalamus and thalamus. Although much is knownabout these structures and the functions that they perform, very littleis understood about the mechanisms that direct their specification,morphogenesis and differentiation. Recently, however, families ofcandidate regulatory genes with regionally restricted expression in theneuroepithelium of the forebrain have been identified; these genefamilies are hypothesized to establish positional identity and tocontrol region-specific morphogenesis and histogenesis of the forebrain(Shimamura et al. 1995 Development, 121:3923-3933; Rubenstein, et al.,1994 Science 266:578-581).

Nkx-2.1 and Nkx-2.2 are two of the earliest known genes to be expressedin the neuroectodermal cells of the forebrain; they are expressed at theonset of neurulation in restricted ventral forebrain domains (Shimamuraet al., 1995 supra). In addition, expression of these genes is inducedby the secreted molecule sonic hedgehog (Shh), a known axialmesendodermal signaling protein that is responsible for the induction ofthe ventral neurons of the forebrain (Ericson et al., 1995 Cell81:747-756; Barth and Wilson, 1995, Development 121:1755-1768). Theearly and spatially limited expression of Nkx-2.1 and Nkx-2.2 inresponse to a primary inductive signal suggests that these moleculesprovide the initial positional information for specific ventral regionsof the developing forebrain.

Nkx-2.2 is a member of a vertebrate gene family that is homologous tothe Drosophila NK-2 gene, which is expressed in neuroblast precursors inthe Drosophila head (Kim and Nirenberg, 1989 Proc. Natl. Acad. Sci. USA.86:7716-7720). The NK-2 gene family is characterized by two regions ofhomology: the homeobox and a highly conserved sequence downstream of thehomeobox.

In addition to its expression in the brain, Nkx-2.2 is also expressed inthe pancreas, pancreatic islet β cells, and hamster insulinoma (HIT)cells (Rudnick et al. 1994 Proc. Natl. Acad. Sci. USA 91:12203-12207).Nkx-2.2 expression in pancreas is accompanied by expression of Nkx-6.1,another NK-2-related gene (Rudnick et al. supra). A partial sequence(Price et al. 1992 Neuron 8:241-255) and a full-length sequence ofmurine Nkx-2.2 (Hartigan and Rubenstein 1996 Gene 168:271-2) have beenpublished. The actual function of Nkx-2.2 or Nkx-6.1 as eithertranscription factors or as developmental regulators was not previouslyknown.

The pathogenesis of diabetes, depression, and obesity, and the linksbetween these diseases, are complex and not well understood. Moreover,the complex nature of these disorders makes their study difficult. Thus,there is a need for an in vivo model for insulin- andserotonin-producing cells for identification of new compounds fortreatment of disorders associated with insulin and serotonin production,as well as for development of new therapies to address such disorders(e.g., methods for replacing or enhancing serotonin-producing and/orinsulin-producing cells). In addition, there is a need for a method toidentify individuals at risk of developing insulin and serotoninproduction-associated disorders. Finally, there is little known aboutthe development and differentiation of the pancreatic islet cells or keycell types in the central nervous system.

SUMMARY OF THE INVENTION

The present invention features a human Nkx-2.2 polypeptide andnucleotide sequences encoding Nkx-2.2 polypeptides. In a particularaspect, the polynucleotide is the nucleotide sequence of SEQ ID NO:1. Inaddition, the invention features polynucleotide sequences that hybridizeunder stringent conditions to SEQ ID NO:1. In related aspects theinvention features expression vectors and host cells comprisingpolynucleotides that encode a human Nkx-2.2 polypeptide. The presentinvention also relates to antibodies that bind specifically to a humanNkx-2.2 polypeptide, and methods for producing human Nkx-2.2polypeptides.

In one aspect the invention features a method for identifying compoundsthat bind a human Nkx-2.2 polypeptide.

Yet another aspect of the invention relates to use of human Nkx-2.2polypeptides and specific antibodies thereto for the diagnosis andtreatment of human disease.

A primary object of the invention is to provide an isolated humanNkx-2.2 polypeptide-encoding polynucleotide for use in expression ofhuman Nkx-2.2 (e.g, in a recombinant host cell) and for use in, forexample, identification of human Nkx-2.2 polypeptide binding compounds(especially those compounds that affect human Nkx-2.2polypeptide-mediated activity).

Another object of the invention is to provide an isolated human Nkx-2.2polypeptide-encoding polynucleotide for use in generation of non-humantransgenic animal models for Nkx-2.2 gene function, wherein thetransgenic animal is characterized by having a defect in Nkx-2.2 genefunction (and, because Nkx-2.2 acts upstream of Nkx-6.1, a defect inNkx-6.1 gene function), and by having a decreased number ofinsulin-producing cells relative to a normal animal of the same species.Such Nkx-2.2 transgenic animals are further characterized by a decreasednumber of serotonin-producing cells relative to a normal animal of thesame species. Another related object of the invention is to providenon-human transgenic mammals that are characterized by excess or ectopicexpression of the Nkx-2.2 gene.

These and other objects, advantages and features of the presentinvention will become apparent to those persons skilled in the art uponreading the details of the invention more fully set forth below.

The invention will now be described in further detail.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B shows the genomic sequence of human Nkx-2.2 polypetide.

DETAILED DESCRIPTION OF THE INVENTION

Before the present nucleotide and polypeptide sequences are described,it is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors and reagentsdescribed as such may, of course, vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “ahost cell” includes a plurality of such host cells and reference to “theantibody” includes reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing, for example, the celllines, vectors, and methodologies which are described in thepublications which might be used in connection with the presentlydescribed invention. The publications discussed herein are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior invention.

Definitions

“Polynucleotide” as used herein refers to an oligonucleotide,nucleotide, and fragments or portions thereof, as well as to peptidenucleic acids (PNA), fragments, portions or antisense molecules thereof,and to DNA or RNA of genomic or synthetic origin which can be single- ordouble-stranded, and represent the sense or antisense strand. Where“polynucleotide” is used to refer to a specific polynucleotide sequence(e.g. a Nkx-2.2 polypeptide-encoding polynucleotide), “polynucleotide”is meant to encompass polynucleotides that encode a polypeptide that isfunctionally equivalent to the recited polypeptide, e.g.,polynucleotides that are degenerate variants, or polynucleotides thatencode biologically active variants or fragments of the recitedpolypeptide. Similarly, “polypeptide” as used herein refers to anoligopeptide, peptide, or protein. Where “polypeptide” is recited hereinto refer to an amino acid sequence of a naturally-occurring proteinmolecule, “polypeptide” and like terms are not meant to limit the aminoacid sequence to the complete, native amino acid sequence associatedwith the recited protein molecule.

By “antisense polynucleotide” is mean a polynucleotide having anucleotide sequence complementary to a given polynucleotide sequence(e.g, a polynucleotide sequence encoding an Nkx-2.2 polypeptide)including polynucleotide sequences associated with the transcription ortranslation of the given polynucleotide sequence (e.g, a promoter of apolynucleotide encoding an Nkx-2.2 polypeptide), where the antisensepolynucleotide is capable of hybridizing to an Nkx-2.2polypeptide-encoding polynucleotide sequence. Of particular interest areantisense polynucleotides capable of inhibiting transcription and/ortranslation of an Nkx-2.2-encoding polynucleotide either in vitro or invivo.

“Peptide nucleic acid” as used herein refers to a molecule whichcomprises an oligomer to which an amino acid residue, such as lysine,and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary (template) strand of nucleic acid (Nielsen et al1993 Anticancer Drug Des 8:53-63).

As used herein, “Nkx-2.2 polypeptide” refers to an amino acid sequenceof a recombinant or nonrecombinant polypeptide having an amino acidsequence of i) a native Nkx-2.2 polypeptide, ii) a biologically activefragment of an Nkx-2.2 polypeptide, iii) biologically active polypeptideanalogs of an Nkx-2.2 polypeptide, or iv) a biologically active variantof an Nkx-2.2 polypeptide. Nkx-2.2 polypeptides of the invention can beobtained from any species, particularly mammalian, including human,rodenti (e.g., murine or rat), bovine, ovine, porcine, murine, orequine, preferably rat or human, from any source whether natural,synthetic, semi-synthetic or recombinant. “Human Nkx-2.2 polypeptide”refers to the amino acid sequences of isolated human Nkx-2.2 polypeptideobtained from a human, and is meant to include all naturally-occurringallelic variants, and is not meant to limit the amino acid sequence tothe complete, native amino acid sequence associated with the recitedprotein molecule.

As used herein, “antigenic amino acid sequence” means an amino acidsequence that, either alone or in association with a carrier molecule,can elicit an antibody response in a mammal.

A “variant” of a human Nkx-2.2 polypeptide is defined as an amino acidsequence that is altered by one or more amino acids. The variant canhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant can have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Similar minorvariations can also include amino acid deletions or insertions, or both.Guidance in determining which and how many amino acid residues may besubstituted, inserted or deleted without abolishing biological orimmunological activity can be found using computer programs well knownin the art, for example, DNAStar software.

A “deletion” is defined as a change in either amino acid or nucleotidesequence in which one or more amino acid or nucleotide residues,respectively, are absent as compared to an amino acid sequence ornucleotide sequence of a naturally occurring Nkx-2.2 polypeptide.

An “insertion” or “addition” is that change in an amino acid ornucleotide sequence which has resulted in the addition of one or moreamino acid or nucleotide residues, respectively, as compared to an aminoacid sequence or nucleotide sequence of a naturally occurring Nkx-2.2.polypeptide.

A “substitution” results from the replacement of one or more amino acidsor nucleotides by different amino acids or nucleotides, respectively ascompared to an amino acid sequence or nucleotide sequence of a naturallyoccurring Nkx-2.2 polypeptide.

“Nkx-2.2 exon 1 polypeptide” and “Nkx-2.2 exon 2 polypeptide” are meantto refer to the amino acid sequences of an isolated polypeptide encodedby the Nkx-2.2 exon 1 and exon 2 sequences, respectively, whichsequences may be obtained from any species, particularly mammalian,including human, rodentia (e.g., murine or rat), bovine, ovine, porcine,murine, or equine, preferably rat or human, from any source whethernatural, synthetic, semi-synthetic or recombinant. “Human Nkx-2.2 exon 1polypeptide” and “human Nkx-2.2 exon 2 polypeptide” refer to the aminoacid sequences of isolated human Nkx-2.2 exon 1 polypeptide and Nkx-2.2exon 2 polypeptide, respectively, obtained from a human, and is meant toinclude all naturally-occurring allelic variants, and is not meant tolimit the amino acid sequence to the complete, native amino acidsequence associated with the recited protein molecule. An exemplaryhuman Nkx-2.2 exon 1 polypeptide is SEQ ID NO:4; an exemplary humanNkx-2.2 exon 2 polypeptide is SEQ ID NO:6.

The term “biologically active” refers to human Nkx-2.2 polypeptidehaving structural, regulatory, or biochemical functions of a naturallyoccurring Nkx-2.2 polypeptide. Likewise, “immunologically active”defines the capability of the natural, recombinant or synthetic humanNkx-2.2 polypeptide, or any oligopeptide thereof, to induce a specificimmune response in appropriate animals or cells and to bind withspecific antibodies.

The term “derivative” as used herein refers to the chemical modificationof a nucleic acid encoding a human Nkx-2.2 polypeptide or the encodedhuman Nkx-2.2 polypeptide. Illustrative of such modifications would bereplacement of hydrogen by an alkyl, acyl, or amino group. A nucleicacid derivative would encode a polypeptide which retains essentialbiological characteristics of a natural Nkx-2.2 polypeptide.

As used herein the term “isolated” is meant to describe a compound ofinterest (e.g., either a polynucleotide or a polypeptide) that is in anenvironment different from that in which the compound naturally occurs.“Isolated” is meant to include compounds that are within samples thatare substantially enriched for the compound of interest and/or in whichthe compound of interest is partially or substantially purified.

As used herein, the term “substantially purified” refers to a compound(e.g., either a polynucleotide or a polypeptide) that is removed fromits natural environment and is at least 60% free, preferably 75% free,and most preferably 90% free from other components with which it isnaturally associated.

“Stringency” typically occurs in a range from about Tm-5° C. (5° C.below the Tm of the probe) to about 20° C. to 25° C. below Tm. As willbe understood by those of skill in the art, a stringency hybridizationcan be used to identify or detect identical polynucleotide sequences orto identify or detect similar or related polynucleotide sequences.

The term “hybridization” as used herein shall include “any process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” (Coombs 1994 Dictionary of Biotechnology, Stockton Press,New York N.Y.). Amplification as carried out in the polymerase chainreaction technologies is described in Dieffenbach et al. 1995, PCRPrimer, a Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.

By “transformation” is meant a permanent or transient genetic change,preferably a permanent genetic change, induced in a cell followingincorporation of new DNA (i.e., DNA exogenous to the cell). Geneticchange can be accomplished either by incorporation of the new DNA intothe genome of the host cell, or by transient or stable maintenance ofthe new DNA as an episomal element. Where the cell is a mammalian cell,a permanent genetic change is generally achieved by introduction of theDNA into the genome of the cell.

By “construct” is meant a recombinant nucleic acid, generallyrecombinant DNA, that has been generated for the purpose of theexpression of a specific nucleotide sequence(s), or is to be used in theconstruction of other recombinant nucleotide sequences.

By “operably linked” is meant that a DNA sequence and a regulatorysequence(s) are connected in such a way as to permit gene expressionwhen the appropriate molecules (e.g., transcriptional activatorproteins) are bound to the regulatory sequence(s).

By “operatively inserted” is meant that a nucleotide sequence ofinterest is positioned adjacent a nucleotide sequence that directstranscription and translation of the introduced nucleotide sequence ofinterest (i.e., facilitates the production of, e.g., a polypeptideencoded by an Nkx-2.2 sequence).

By “Nkx-2.2 associated disorder” is meant a physiological condition ordisease associated with altered Nkx-2.2 function (e.g., due to aberrantNkx-2.2 expression or a defect in Nkx-2.2 expression or in the Nkx-2.2protein). Such Nkx-2.2 associated disorders can include, but are notnecessarily limited to, disorders associated with reduced levels ofinsulin or the ability to utilize insulin (e.g., hyperglycemia, diabetes(e.g., Type 1 and Type 2 diabetes, and the like), Parkinson's, disordersassociated with reduced serotonin production (e.g,. depression andobesity), and disorders associated with neural defects (e.g., defects inmotor neurons, serotonin-producing neurons, dopamine neurons, anddevelopmental defects in the forebrain, midbrain, hindbrain, and spinalcord).

By “Nkx-6.1 associated disorder” is meant a physiological condition ordisease associated with altered Nkx-6.1 function (e.g., due to aberrantNkx-6.1 expression or a defect in Nkx-6.1 expression or in the Nkx-6.1protein). Such Nkx-6.1 associated disorders can include, but are notnecessarily limited to, disorders associated with reduced levels ofinsulin or the ability to utilize insulin (e.g., hyperglycemia, diabetes(e.g., Type 1 and Type 2 diabetes, and the like), Parkinson's, anddisorders associated with neural defects (e.g., defects in motorneurons, serotonin-producing neurons, dopamine neurons, anddevelopmental defects in the forebrain, midbrain, hindbrain, and spinalcord).

By “subject” or “patient” is meant any subject for which therapy isdesired, including humans, cattle, dogs, cats, guinea pigs, rabbits,rats, mice, insects, horses, chickens, and so on. Of particular interestare subjects having an Nkx-2.2-associated disorder which is amenable totreatment (e.g., to mitigate symptoms associated with the disorder) byexpression of either Nkx-2.2-encoding nucleic acid in a cell of thesubject (e.g., by introduction of the Nkx-2.2-encoding nucleic acid intothe subject in vivo, or by implanting Nkx-2.2-expressing cells into thesubject, which cells also produce a hormone that the subject is in needof (e.g., insulin or serotonin)).

The term “transgene” is used herein to describe genetic material whichhas been or is about to be artificially inserted into the genome of amammalian, particularly a mammalian cell of a living animal.

By “transgenic organism” is meant a non-human organism (e.g.,single-cell organisms (e.g., yeast), mammal, non-mammal (e.g., nematodeor Drosophila)) having a non-endogenous (i.e., heterologous) nucleicacid sequence present as an extrachromosomal element in a portion of itscells or stably integrated into its germ line DNA.

By “transgenic animal” is meant a non-human animal, usually a mammal,having a non-endogenous (i.e., heterologous) nucleic acid sequencepresent as an extrachromosomal element in a portion of its cells orstably integrated into its germ line DNA (i.e., in the genomic sequenceof most or all of its cells). Heterologous nucleic acid is introducedinto the germ line of such transgenic animals by genetic manipulationof, for example, embryos or embryonic stem cells of the host animal.

A “knock-out” of a target gene means an alteration in the sequence ofthe gene that results in a decrease of function of the target gene,preferably such that target gene expression is undetectable orinsignificant. A knock-out of an Nkx-2.2 gene or Nkx-6.1 gene means thatfunction of the Nkx-2.2 gene or Nkx-6.1 gene, respectively, has beensubstantially decreased so that Nkx-2.2 or Nkx-6.1 expression is notdetectable or only present at insignificant levels. “Knock-out”transgenics of the invention can be transgenic animals having aheterozygous knock-out of the Nkx-2.2 gene, a homozygous knock-out ofthe Nkx-2.2 gene, heterozygous knock-out of the Nkx-6.1 gene, ahomozygous knock-out of the Nkx-6.1 gene, or any combination thereof.“Knock-outs” also include conditional knock-outs, where alteration ofthe target gene can occur upon, for example, exposure of the animal to asubstance that promotes target gene alteration, introduction of anenzyme that promotes recombination at the target gene site (e.g., Cre inthe Cre-lox system), or other method for directing the target genealteration postnatally.

A “knock-in” of a target gene means an alteration in a host cell genomethat results in altered expression (e.g., increased (including ectopic)or decreased expression) of the target gene, e.g., by introduction of anadditional copy of the target gene, or by operatively inserting aregulatory sequence that provides for enhanced expression of anendogenous copy of the target gene. “Knock-in” transgenics of theinvention can be transgenic animals having a heterozygous knock-in ofthe Nkx-2.2 gene, a homozygous knock-in of the Nkx-2.2 gene,heterozygous knock-in of the Nkx-6.1 gene, a homozygous knock-in of theNkx-6.1 gene, or any combination thereof. “Knock-ins” also encompassconditional knock-ins.

Overview of the Invention

The present invention is based upon the identification and isolation ofa polynucleotide sequence encoding a human Nkx-2.2 polypeptide.Accordingly, the present invention encompasses such human Nkx-2.2polypeptide-encoding polynucleotides, as well as human Nkx-2.2polypeptides encoded by such polynucleotides. Expression of Nkx-2.2 islinked to both pancreatic and neural development. Specifically, Nkx-2.2expression is necessary for development of β cells, the cellsresponsible for insulin production that are located in the islets ofLangerhans in the pancreas. Furthermore, Nkx-2.2 expression is alsoessential for development of serotonin-secreting cells in the brain.

The invention also encompasses the use of the polynucleotides disclosedherein to facilitate identification and isolation of polynucleotide andpolypeptide sequences having homology to a human Nkx-2.2 polypeptide ofthe invention. The human Nkx-2.2 polypeptides and polynucleotides of theinvention are also useful in the identification of human Nkx-2.2polypeptide-binding compounds, particularly human Nkx-2.2polypeptide-binding compounds having human Nkx-2.2 polypeptide agonistor antagonist activity. In addition, the human Nkx-2.2 polypeptides andpolynucleotides of the invention are useful in the diagnosis, preventionand treatment of disease associated with human Nkx-2.2 polypeptidebiological activity.

The human Nkx-2.2 polypeptide-encoding polynucleotides of the inventioncan also be used as a molecular probe with which to determine thestructure, location, and expression of the human Nkx-2.2 polypeptide andrelated polypeptides in mammals (including humans) and to investigatepotential associations between disease states or clinical disorders anddefects or alterations in human Nkx-2.2 polypeptide structure,expression, or function.

Nkx-2.2 Nucleic Acid

The term “Nkx-2.2 gene” is used generically to designate Nkx-2.2 genesand their alternate forms. “Nkx-2.2 gene” is also intended to mean theopen reading frame encoding specific Nkx-2.2 polypeptides, introns, andadjacent 5′ and 3′ non-coding nucleotide sequences involved in theregulation of expression, up to about 1 kb beyond the coding region, butpossibly further in either direction. The DNA sequences encoding Nkx-2.2may be cDNA or genomic DNA or a fragment thereof. The gene may beintroduced into an appropriate vector for extrachromosomal maintenanceor for integration into the host.

The term “cDNA” as used herein is intended to include all nucleic acidsthat share the arrangement of sequence elements found in native maturemRNA species, where sequence elements are exons and 3′ and 5′ non-codingregions. Normally mRNA species have contiguous exons, with theintervening introns removed by nuclear RNA splicing, to create acontinuous open reading frame encoding the Nkx-2.2 polypeptide.

Genomic Nkx-2.2 sequences have non-contiguous open reading frames, whereintrons interrupt the protein coding regions. A genomic sequence ofinterest comprises the nucleic acid present between the initiation codonand the stop codon, as defined in the listed sequences, including all ofthe introns that are normally present in a native chromosome. It mayfurther include the 3′ and 5′ untranslated regions found in the maturemRNA. It may further include specific transcriptional and translationalregulatory sequences, such as promoters, enhancers, etc., includingabout 1 kb, but possibly more, of flanking genomic DNA at either the 5′or 3′ end of the transcribed region. The genomic DNA may be isolated asa fragment of 100 kbp or smaller; and substantially free of flankingchromosomal sequence.

The sequence of this 5′ region, and further 5′ upstream sequences and 3′downstream sequences, may be utilized for promoter elements, includingenhancer binding sites, that provide for expression in tissues whereNkx-2.2 is expressed. The tissue specific expression is useful fordetermining the pattern of expression, and for providing promoters thatmimic the native pattern of expression. Naturally occurringpolymorphisms in the promoter region are useful for determining naturalvariations in expression, particularly those that may be associated withdisease. Alternatively, mutations may be introduced into the promoterregion to determine the effect of altering expression in experimentallydefined systems. Methods for the identification of specific DNA motifsinvolved in the binding of transcriptional factors are known in the art,e.g. sequence similarity to known binding motifs, gel retardationstudies, etc. For examples, see Blackwell et al. 1995 Mol Med 1:194-205;Mortlock et al. 1996 Genome Res. 6: 327-33; and Joulin and Richard-Foy(1995) Eur J Biochem 232: 620-626.

The regulatory sequences may be used to identify cis acting sequencesrequired for transcriptional or translational regulation of Nkx-2.2expression, especially in different tissues or stages of development,and to identify cis acting sequences and trans acting factors thatregulate or mediate Nkx-2.2 expression. Such transcriptional ortranslational control regions may be operably linked to an Nkx-2.2 geneor other genes in order to promote expression of wild type or alteredNkx-2.2 or other proteins of interest in cultured cells, or inembryonic, fetal or adult tissues, and for gene therapy.

The nucleic acid compositions used in the subject invention may encodeall or a part of the Nkx-2.2 polypeptides as appropriate. Fragments maybe obtained of the DNA sequence by chemically synthesizingoligonucleotides in accordance with conventional methods, by restrictionenzyme digestion, by PCR amplification, etc. For the most part, DNAfragments will be of at least about ten contiguous nucleotides, usuallyat least about 15 nt, more usually at least about 18 nt to about 20 nt,more usually at least about 25 nt to about 50 nt. Such small DNAfragments are useful as primers for PCR, hybridization screening, etc.Larger DNA fragments, i.e. greater than 100 nt are useful for productionof the encoded polypeptide. For use in amplification reactions, such asPCR, a pair of primers will be used. The exact composition of the primersequences is not critical to the invention, but for most applicationsthe primers will hybridize to the subject sequence under stringentconditions, as known in the art. It is preferable to choose a pair ofprimers that will generate an amplification product of at least about 50nt, preferably at least about 100 nt. Algorithms for the selection ofprimer sequences are generally known, and are available in commercialsoftware packages. Amplification primers hybridize to complementarystrands of DNA, and will prime towards each other.

The Nkx-2.2 gene is isolated and obtained in substantial purity,generally as other than an intact mammalian chromosome. Usually, the DNAwill be obtained substantially free of other nucleic acid sequences thatdo not include an Nkx-2.2. sequence or fragment thereof, generally beingat least about 50%, usually at least about 90% pure and are typically“recombinant”, i.e. flanked by one or more nucleotides with which it isnot normally associated on a naturally occurring chromosome.

The DNA sequences are used in a variety of ways. They may be used asprobes for identifying homologs of Nkx-2.2. Mammalian homologs havesubstantial sequence similarity to one another, i.e. at least 75%,usually at least 90%, more usually at least 95% sequence identity.Sequence similarity is calculated based on a reference sequence, whichmay be a subset of a larger sequence, such as a conserved motif, codingregion, flanking region, etc. A reference sequence will usually be atleast about 18 nt long, more usually at least about 30 nt long, and mayextend to the complete sequence that is being compared. Algorithms forsequence analysis are known in the art, such as BLAST, described inAltschul et al. 1990 J Mol Biol 215:403-10.

Nucleic acids having sequence similarity are detected by hybridizationunder low stringency conditions, for example, at 50° C. and 6XSSC (0.9 Msaline/0.09 M sodium citrate) and remain bound when subjected to washingat 55° C. in 1XSSC (0.15 M sodium chloride/0.015 M sodium citrate).Sequence identity may be determined by hybridization under highstringency conditions, for example, at 50° C. or higher and 0.1XSSC (15mM saline/0.15 mM sodium citrate). By using probes, particularly labeledprobes of DNA sequences, one can isolate homologous or related genes.The source of homologous genes may be any species, e.g. primate species,particularly human; rodents, such as rats and mice, canines, felines,bovines, ovines, equines, yeast, Drosophila, Caenhorabditis, etc.

The Nkx-2.2-encoding DNA may also be used to identify expression of thegene in a biological specimen. The manner in which one probes cells forthe presence of particular nucleotide sequences, as genomic DNA or RNA,is well established in the literature and does not require elaborationhere. mRNA is isolated from a cell sample. mRNA may be amplified byRT-PCR, using reverse transcriptase to form a complementary DNA strand,followed by polymerase chain reaction amplification using primersspecific for the subject DNA sequences. Alternatively, mRNA sample isseparated by gel electrophoresis, transferred to a suitable support,e.g. nitrocellulose, nylon, etc., and then probed with a fragment of thesubject DNA as a probe. Other techniques, such as oligonucleotideligation assays, in situ hybridizations, and hybridization to DNA probesarrayed on a solid chip may also find use. Detection of mRNA hybridizingto an Nkx-2.2 sequence is indicative of Nkx-2.2 gene expression in thesample.

The Nkx-2.2 nucleic acid sequence may be modified for a number ofpurposes, particularly where they will be used intracellularly, forexample, by being joined to a nucleic acid cleaving agent, e.g. achelated metal ion, such as iron or chromium for cleavage of the gene;or the like.

The sequence of the Nkx-2.2 locus, including flanking promoter regionsand coding regions, may be mutated in various ways known in the art togenerate targeted changes in promoter strength, sequence of the encodedprotein, etc. The DNA sequence or product of such a mutation will besubstantially similar to the sequences provided herein, i.e. will differby at least one nucleotide or amino acid, respectively, and may differby at least two but not more than about ten nucleotides or amino acids.The sequence changes may be substitutions, insertions or deletions.Deletions may further include larger changes, such as deletions of adomain or exon. Other modifications of interest include epitope tagging,e.g. with the FLAG system, HA, etc. For studies of subcellularlocalization, fusion proteins with green fluorescent proteins (GFP) maybe used. Such mutated genes may be used to study structure-functionrelationships of Nkx-2.2 polypeptides with other polypeptides (e.g.,Nkx-6.1), or to alter properties of the proteins that affect theirfunction or regulation. Such modified Nkx-2.2 sequences can be used to,for example, generate the transgenic animals.

Techniques for in vitro mutagenesis of cloned genes are known. Examplesof protocols for scanning mutations may be found in Gustin et al., 1993Biotechniques 14:22; Barany, 1985 Gene 37:111-23; Colicelli et al., 1985Mol Gen Genet 199:537-9; and Prentki et al., 1984 Gene 29:303-13.Methods for site specific mutagenesis can be found in Sambrook et al.,1989 Molecular Cloning: A Laboratory Manual, CSH Press, pp. 15.3-15.108;Weiner et al., 1993 Gene 126:35-41; Sayers et al., 1992 Biotechniques13:592-6; Jones and Winistorfer, 1992 Biotechniques 12:528-30; Barton etal., 1990 Nucleic Acids Res 18:7349-55; Marotti and Tomich, 1989 GeneAnal Tech 6:67-70; and Zhu 1989 Anal Biochem 177:120-4.

Nkx-2.2 Transgenic Animals

The Nkx-2.2-encoding nucleic acids can be used to generate geneticallymodified non-human animals or site specific gene modifications in celllines. The term “transgenic” is intended to encompass geneticallymodified animals having a deletion or other knock-out of Nkx-2.2 geneactivity, having an exogenous Nkx-2.2 gene that is stably transmitted inthe host cells, “knock-in” having altered Nkx-2.2 gene expression, orhaving an exogenous Nkx-2.2 promoter operably linked to a reporter gene.Of particular interest are homozygous and heterozygous knock-outs ofNkx-2.2. Transgenics that are homozygous knock-outs for Nkx-2.2 arelikely of less interest due to the lethality of this combination.Transgenics that are heterozygous knock-outs for Nkx-2.2 may besusceptible to an Nkx-2.2-associated disorder (e.g,. diabetes, obesity)at a later age.

Transgenic animals may be made through homologous recombination, wherethe Nkx-2.2 locus is altered. Alternatively, a nucleic acid construct israndomly integrated into the genome. Vectors for stable integrationinclude plasmids, retroviruses and other animal viruses, YACs, and thelike. Of interest are transgenic mammals, preferably a mammal from agenus selected from the group consisting of Mus (e.g., mice), Rattus(e.g., rats), Oryctologus (e.g., rabbits) and Mesocricetus (e.g.,hamsters). More preferably the animal is a mouse which is defective orcontains some other alteration in Nkx-2.2 gene expression or function.Without being held to theory, within the pancreas Nkx-2.2 apparentlyacts as a transcriptional activator in a cascade pathway upstream of thetranscriptional activity of Nkx-6.1. Therefore, alteration of Nkx-2.2function will affect Nkx-6.1 function (e.g., a Nkx-2.2 knock-outtransgenic animal will have no or little Nkx-6.1 activity present incells normally exhibiting such activity).

A “knock-out” animal is genetically manipulated to substantially reduce,or eliminate endogenous Nkx-2.2 function, preferably such that targetgene expression is undetectable or insignificant. Different approachesmay be used to achieve the “knock-out”. A chromosomal deletion of all orpart of the native Nkx-2.2 homolog may be induced. Deletions of thenon-coding regions, particularly the promoter region, 3′ regulatorysequences, enhancers, or deletions of gene that activate expression ofthe Nkx-2.2 genes. A functional knock-out may also be achieved by theintroduction of an anti-sense construct that blocks expression of thenative Nkx-2.2 gene (for example, see Li and Cohen (1996) Cell85:319-329).

Homozygous knock-outs of the endogenous Nkx-2.2 gene results in adramatic decrease in insulin production as well as severe neuraldefects. Because transgenic animals having a homozygous knock-out of theNkx-2.2 gene do not survive long after birth (e.g, null Nkx-2.2 micesurvive no more than a few days postnatally (e.g., from about 3 days toabout 6 days), use of transgenic animals heterozygous for an Nkx-2.2gene knock-out, or transgenic animals homozygous for a less severedefect in Nkx-2.2 (e.g., a defect that is not lethal within a few hoursto days after birth) may be useful as well. For example, a defect inNkx-2.2 function is associated with a decrease in glucokinase expressionin the pancreas in homozygous Nkx-2.2 knock-outs (see Examples below).Since glucokinase is the rate-limiting step in β cell glucose sensing,even modest reductions in glucokinase expression due to altered Nkx-2.2.expression (e.g., due to a heterozygous defect in Nkx-2.2) coulddecrease β cell glucose sensitivity and causes inadequate insulinproduction and secretion. Moreover, homozygous null Nkx-2.2 transgenicshad roughly normal islet amyloid polypeptide (amylin) expression (seethe Examples below). Since amyloid deposits of this peptide have beenproposed to cause β cell damage and progressive loss of insulinproduction in type 2 diabetes, a decreased ratio of insulin/amylinproduction in individuals with decreased Nkx-2.2 may be anothercontributor to the disease.

Conditional knock-outs of Nkx-2.2 gene function can also be generated.Conditional knock-outs are transgenic animals that exhibit a defect inNkx-2.2 gene function upon exposure of the animal to a substance thatpromotes target gene alteration, introduction of an enzyme that promotesrecombination at the target gene site (e.g., Cre in the Cre-loxPsystem), or other method for directing the target gene alteration.

For example, a transgenic animal having a conditional knock-out ofNkx-2.2 gene function can be produced using the Cre-loxP recombinationsystem (see, e.g., Kilby et al. 1993 Trends Genet 9:413-421). Cre is anenzyme that excises the DNA between two recognition sequences, termedloxP. This system can be used in a variety of ways to create conditionalknock-outs of Nkx-2.2. For example, two independent transgenic mice canbe produced: one transgenic for an Nkx-2.2. sequence flanked by loxPsites and a second transgenic for Cre. The Cre transgene can be underthe control of an inducible or developmentally regulated promoter (Gu etal. 1993 Cell 73:1155-1164; Gu et al. 1994 Science 265:103-106), orunder control of a tissue-specific or cell type-specific promoter (e.g.,a pancreas-specific promoter or brain tissue-specific promoter). TheNkx-2.2 transgenic is then crossed with the Cre transgenic to produceprogeny deficient for the Nkx-2.2 gene only in those cells thatexpressed Cre during development.

Transgenic animals may be made having an exogenous Nkx-2.2 gene. Forexample, the transgenic animal may comprise a “knock-in” of an Nkx-2.2gene, such that the host cell genome contains an alteration that resultsin altered expression (e.g., increased (including ectopic) or decreasedexpression) of an Nkx-2.2 gene, e.g., by introduction of an additionalcopy of the target gene, or by operatively inserting a regulatorysequence that provides for enhanced expression of an endogenous copy ofthe target gene. “Knock-in” transgenics can be transgenic animals havinga heterozygous knock-in of the Nkx-2.2 gene or a homozygous knock-in ofthe Nkx-2.2. “Knock-ins” also encompass conditional knock-ins.

The exogenous gene introduced into the host cell genome to produce atransgenic animal is usually either from a different species than theanimal host, or is otherwise altered in its coding or non-codingsequence. The introduced gene may be a wild-type gene, naturallyoccurring polymorphism, or a genetically manipulated sequence, forexample those previously described with deletions, substitutions orinsertions in the coding or non-coding regions. The introduced sequencemay encode an Nkx-2.2 polypeptide, or may utilize the Nkx-2.2 promoteroperably linked to a reporter gene. Where the introduced gene is acoding sequence, it is usually operably linked to a promoter, which maybe constitutive or inducible, and other regulatory sequences requiredfor expression in the host animal.

Specific constructs of interest include, but are not limited to,anti-sense Nkx-2.2, or a ribozyme based on an Nkx-2.2 sequence, whichwill block Nkx-2.2 expression, as well as expression of dominantnegative Nkx-2.2 mutations, and over-expression of an Nkx-2.2 gene. Adetectable marker, such as lac Z may be introduced into the Nkx-2.2locus, where upregulation of expression of the corresponding Nkx genewill result in an easily detected change in phenotype. Constructsutilizing a promoter region of the Nkx-2.2 genes in combination with areporter gene or with the coding region of Nkx-2.2 are also of interest.Constructs having a sequence encoding a truncated or altered (e.g,mutated) Nkx-2.2 are also of interest.

The modified cells or animals are useful in the study of function andregulation of Nkx-2.2 and, since Nkx-2.2 is thought to act upstream ofNkx-6.1, of Nkx-6.1. Such modified cells or animals are also useful in,for example, the study of the function and regulation of genes whoseexpression is affected by Nkx-2.2, as well as the study of thedevelopment of insulin-secreting cells in the pancreas, and thedevelopment of serotonin-secreting cells in the brain. Thus, thetransgenic animals of the invention are useful in identifying downstreamtargets of Nkx-2.2, as such targets may have a role in the phenotypesassociated with defects in Nkx-2.2.

Animals may also be used in functional studies, drug screening, etc.,e.g. to determine the effect of a candidate drug on islet celldevelopment, on β-cell function and development, on serotonin-secretingcell development, or on symptoms associated with disease or conditionsassociated with Nkx-2.2 defects (e.g., on symptoms associated withreduced insulin secretion (e.g., such as that associated with a diabeticsyndrome, including Type 2 diabetes), symptoms associated with obesity,or on symptoms associated with reduced serotonin secretion (e.g,symptoms associated with depression). A series of small deletions and/orsubstitutions may be made in the Nkx-2.2 genes to determine the role ofdifferent exons in DNA binding, transcriptional regulation, etc. Byproviding expression of Nkx-2.2 protein in cells in which it isotherwise not normally produced (e.g., ectopic expression), one caninduce changes in cell behavior. These animals are also useful forexploring models of inheritance of disorders associated with depression,obesity, and/or diabetes, e.g. dominant v. recessive; relative effectsof different alleles and synergistic effects between Nkx-2.2 and othergenes elsewhere in the genome.

DNA constructs for homologous recombination will comprise at least aportion of the Nkx-2.2 gene with the desired genetic modification, andwill include regions of homology to the target locus. DNA constructs forrandom integration need not include regions of homology to mediaterecombination. Conveniently, markers for positive and negative selectionare included. Methods for generating cells having targeted genemodifications through homologous recombination are known in the art. Forvarious techniques for transfecting mammalian cells, see Keown et al.1990 Methods in Enzymology 185:527-537.

For embryonic stem (ES) cells, an ES cell line may be employed, orembryonic cells may be obtained freshly from a host, e.g. mouse, rat,guinea pig, etc. Such cells are grown on an appropriatefibroblast-feeder layer or grown in the presence of appropriate growthfactors, such as leukemia inhibiting factor (LIF). When ES cells havebeen transformed, they may be used to produce transgenic animals. Aftertransformation, the cells are plated onto a feeder layer in anappropriate medium. Cells containing the construct may be detected byemploying a selective medium. After sufficient time for colonies togrow, they are picked and analyzed for the occurrence of homologousrecombination or integration of the construct. Those colonies that arepositive may then be used for embryo manipulation and blastocystinjection. Blastocysts are obtained from 4 to 6 week old superovulatedfemales. The ES cells are trypsinized, and the modified cells areinjected into the blastocoel of the blastocyst. After injection, theblastocysts are returned to each uterine horn of pseudopregnant females.Females are then allowed to go to term and the resulting littersscreened for mutant cells having the construct. By providing for adifferent phenotype of the blastocyst and the ES cells, chimeric progenycan be readily detected.

The chimeric animals are screened for the presence of the modified gene.Chimeric animals having the modification (normally chimeric males) aremated with wildtype animals to produce heterozygotes, and theheterozygotes mated to produce homozygotes. If the gene alterationscause lethality at some point in development, tissues or organs can bemaintained as allogeneic or congenic grafts or transplants, or in invitro culture.

Investigation of genetic function may utilize non-mammalian models,particularly using those organisms that are biologically and geneticallywell-characterized, such as C. elegans, D. melanogaster and S.cerevisiae. For example, transposon (Tc1) insertions in the nematodehomolog of an Nkx-2.2 gene or a promoter region of an Nkx-2.2 gene maybe made. The Nkx-2.2 gene sequences may be used to knock-out or tocomplement defined genetic lesions in order to determine thephysiological and biochemical pathways involved in function of isletcells and/or serotonin-secreting cells. It is well known that humangenes can complement mutations in lower eukaryotic models.

Production of Nkx-2.2 Polypeptides

Nkx-2.2-encoding nucleic acid may be employed to synthesize full-lengthNkx-2.2 polypeptides or fragments thereof, particularly fragmentscorresponding to functional domains; DNA binding sites; etc.; andincluding fusions of the subject polypeptides to other proteins or partsthereof. For expression, an expression cassette may be employed,providing for a transcriptional and translational initiation region,which may be inducible or constitutive, where the coding region isoperably linked under the transcriptional control of the transcriptionalinitiation region, and a transcriptional and translational terminationregion. Various transcriptional initiation regions may be employed thatare functional in the expression host.

The polypeptides may be expressed in prokaryotes or eukaryotes inaccordance with conventional ways, depending upon the purpose forexpression. For large scale production of the protein, a unicellularorganism, such as E. coli, B. subtilis, S. cerevisiae, or cells of ahigher organism such as vertebrates, particularly mammals, e.g. COS 7cells, may be used as the expression host cells. In many situations, itmay be desirable to express the Nkx-2.2 genes in mammalian cells,especially where the encoded polypeptides will benefit from nativefolding and post-translational modifications. Small peptides can also besynthesized in the laboratory.

With the availability of the polypeptides in large amounts, by employingan expression host, the polypeptides may be isolated and purified inaccordance with conventional ways. A lysate may be prepared of theexpression host and the lysate purified using HPLC, exclusionchromatography, gel electrophoresis, affinity chromatography, or otherpurification technique. The purified polypeptide will generally be atleast about 80% pure, preferably at least about 90% pure, and may be upto and including 100% pure. Pure is intended to mean free of otherproteins, as well as cellular debris.

The Nkx-2.2 polypeptides can be used for the production of antibodies,where short fragments provide for antibodies specific for the particularpolypeptide, and larger fragments or the entire protein allow for theproduction of antibodies over the surface of the polypeptide. Antibodiesmay be raised to the wild-type or variant forms of Nkx-2.2. Antibodiesmay be raised to isolated peptides corresponding to these domains, or tothe native protein, e.g. by immunization with cells expressing Nkx-2.2,immunization with liposomes having Nkx-2.2 polypeptides inserted in themembrane, etc.

Antibodies are prepared in accordance with conventional ways, where theexpressed polypeptide or protein is used as an immunogen, by itself orconjugated to known immunogenic carriers, e.g. KLH, pre-S HBsAg, otherviral or eukaryotic proteins, or the like. Various adjuvants may beemployed, with a series of injections, as appropriate. For monoclonalantibodies, after one or more booster injections, the spleen isisolated, the lymphocytes immortalized by cell fusion, and then screenedfor high affinity antibody binding. The immortalized cells, i.e.hybridomas, producing the desired antibodies may then be expanded. Forfurther description, see Monoclonal Antibodies: A Laboratory Manual,Harlow and Lane eds., Cold Spring Harbor Laboratories, Cold SpringHarbor, N.Y., 1988. If desired, the mRNA encoding the heavy and lightchains may be isolated and mutagenized by cloning in E. coli, and theheavy and light chains mixed to further enhance the affinity of theantibody. Alternatives to in vivo immunization as a method of raisingantibodies include binding to phage “display” libraries, usually inconjunction with in vitro affinity maturation.

Isolation of Nkx-2.2 Allelic Variants and Homologues in Other Species

Other mammalian Nkx-2.2 genes can be identified and their functioncharacterized using the Nkx-2.2 genes used in the present invention.Other mammalian Nkx-2.2 genes of interest include, but are not limitedto, human, rodent (e.g, murine, or rat), bovine, feline, canine, and thelike. Methods for identifying, isolating, sequencing, and characterizingan unknown gene based upon its homology to a known gene sequence arewell known in the art (see, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, CSH Press 1989.

Drug Screening

The animal models of the invention, as well as methods using the Nkx-2.2polypeptides in vitro, can be used to identify candidate agents thataffect Nkx-2.2 expression or that interact with Nkx-2.2. polypeptides.Agents of interest can include those that enhance, inhibit, regulate, orotherwise affect Nkx-2.2. activity and/or expression. Agents thatenhance Nkx-2.2 activity and/or expression can be used to treat or studydisorders associated with decreased Nkx-2.2 activity (e.g,. diabetes,obesity, depression), while agents that decrease Nkx-2.2. activityand/or express can be used to treat or study disorders associated withNkx-2.2 activity (e.g, increased activity, e.g, insulinomas, expressionduring development). Candidate agents is meant to include syntheticmolecules (e.g., small molecule drugs, peptides, or other syntheticallyproduced molecules or compounds, as well as recombinantly produced geneproducts) as well as naturally-occurring compounds (e.g., polypeptides,endogenous factors present in insulin-producing and/orserotonin-producing cells, hormones, plant extracts, and the like).

Drug Screening Assays

Of particular interest in the present invention is the identification ofagents that have activity in affecting Nkx-2.2 expression and/orfunction. Such agents are candidates for development of treatments for,for example, diabetes (especially Type 2 diabetes), depression, andobesity (in the case of Nkx-2.2.). Drug screening identifies agents thatprovide a replacement or enhancement for Nkx-2.2 function in affectedcells. Conversely, agents that reverse or inhibit Nkx-2.2 function mayprovide a means to regulate insulin or serotonin production. Ofparticular interest are screening assays for agents that have a lowtoxicity for human cells.

The term “agent” as used herein describes any molecule, e.g. protein orpharmaceutical, with the capability of altering or mimicking thephysiological function of Nkx-2.2. Generally a plurality of assaymixtures are run in parallel with different agent concentrations toobtain a differential response to the various concentrations. Typically,one of these concentrations serves as a negative control, i.e. at zeroconcentration or below the level of detection.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 50 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including, but not limited to: peptides, saccharides, fattyacids, steroids, purines, pyrimidines, derivatives, structural analogsor combinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

Screening of Candidate Agents In Vivo

Agents can be screened for their ability to affect Nkx-2.2. expressionor function or to mitigate an undesirable phenotype (e.g., a symptom)associated with an alteration in Nkx-2.2 expression or function. In apreferred embodiment, screening of candidate agents is performed in vivoin a transgenic animal described herein. Transgenic animals suitable foruse in screening assays include any transgenic animal having analteration in Nkx-2.2 expression, and can include transgenic animalshaving a homozygous or heterozygous knockout of an Nkx-2.2 gene, anexogenous and stably transmitted mammalian Nkx-2.2 gene sequence, and areporter gene composed of an Nkx-2.2 promoter sequence operably linkedto a reporter gene (e.g,. β-galactosidase, CAT, or other gene that canbe easily assayed for expression). The transgenic animals can be eitherhomozygous or heterozygous for the genetic alteration and, where asequence is introduced into the animal's genome for expression, maycontain multiple copies of the introduced sequence.

The candidate agent is administered to a non-human, transgenic animalhaving altered Nkx-2.2 expression, and the effects of the candidateagent determined. The candidate agent can be administered in any mannerdesired and/or appropriate for delivery of the agent in order to effecta desired result. For example, the candidate agent can be administeredby injection (e.g., by injection intravenously, intramuscularly,subcutaneously, or directly into the tissue in which the desired affectis to be achieved), orally, or by any other desirable means. Normally,the in vivo screen will involve a number of animals receiving varyingamounts and concentrations of the candidate agent (from no agent to anamount of agent hat approaches an upper limit of the amount that can bedelivered successfully to the animal), and may include delivery of theagent in different formulation. The agents can be administered singly orcan be combined in combinations of two or more, especially whereadministration of a combination of agents may result in a synergisticeffect.

The effect of agent administration upon the transgenic animal can bemonitored by assessing Nkx-2.2 function as appropriate (e.g., byexamining expression of a reporter or fusion gene), or by assessing aphenotype associated with the Nkx-2.2 expression. For example, where thetransgenic animal used in the screen contains a defect in Nkx-2.2expression (e.g., due to a knock-out of the gene), the effect of thecandidate agent can be assessed by determining levels of hormonesproduced in the mouse relative to the levels produced in the Nkx-2.2defective transgenic mouse and/or in wildtype mice (e.g, by assessinglevels of insulin, serotonin, and/or glucagon). Methods for assayinginsulin, glucagon, and serotonin are well known in the art. Where thecandidate agent affects Nkx-2.2 expression, and/or affects anNkx-2.2-associated phenotype, in a desired manner, the candidate agentis identified as an agent suitable for use in therapy of anNkx-2.2-associated disorder.

Screening of Candidate Agents In Vitro

In addition to screening of agents in Nkx-2.2 transgenic animals, a widevariety of in vitro assays may be used for this purpose, includinglabeled in vitro protein-protein binding assays, protein-DNA bindingassays, electrophoretic mobility shift assays, immunoassays for proteinbinding, and the like. For example, by providing for the production oflarge amounts of Nkx-2.2 protein, one can identify ligands or substratesthat bind to, modulate or mimic the action of the proteins. The purifiedprotein may also be used for determination of three-dimensional crystalstructure, which can be used for modeling intermolecular interactions,transcriptional regulation, etc.

The screening assay can be a binding assay, wherein one or more of themolecules may be joined to a label, and the label directly or indirectlyprovide a detectable signal. Various labels include radioisotopes,fluorescers, chemiluminescers, enzymes, specific binding molecules,particles, e.g. magnetic particles, and the like. Specific bindingmolecules include pairs, such as biotin and streptavidin, digoxin andantidigoxin etc. For the specific binding members, the complementarymember would normally be labeled with a molecule that provides fordetection, in accordance with known procedures.

A variety of other reagents may be included in the screening assaysdescribed herein. Where the assay is a binding assay, these includereagents like salts, neutral proteins, e.g. albumin, detergents, etcthat are used to facilitate optimal protein-protein binding, protein-DNAbinding, and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc. may be used. Themixture of components are added in any order that provides for therequisite binding. Incubations are performed at any suitabletemperature, typically between 4 and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high-throughput screening. Typically between 0.1 and 1 hours willbe sufficient.

Other assays of interest detect agents that mimic Nkx-2.2 function. Forexample, candidate agents are added to a cell that lacks functionalNkx-2.2, and screened for the ability to reproduce Nkx-2.2 activity in afunctional assay.

Many mammalian genes have homologs in yeast and lower animals. The studyof such homologs' physiological role and interactions with otherproteins in vivo or in vitro can facilitate understanding of biologicalfunction. In addition to model systems based on genetic complementation,yeast has been shown to be a powerful tool for studying protein-proteininteractions through the two hybrid system described in Chien et al.1991 Proc. Natl. Acad. Sci. USA 88:9578-9582. Two-hybrid system analysisis of particular interest for exploring transcriptional activation byNkx-2.2 proteins and to identify cDNAs encoding polypeptides thatinteract with Nkx-2.2.

Identified Candidate Agents

The compounds having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a host fortreatment of a condition attributable to a defect in Nkx-2.2 function(e.g., a disorder associated with reduced insulin levels (e.g., diabetes(Type 1 or Type 2 diabetes, particularly Type 1 diabetes); a disorderassociated with reduced serotonin levels (e.g, depression and/orobesity). The compounds may also be used to enhance Nkx-2.2 function.The therapeutic agents may be administered in a variety of ways, orally,topically, parenterally e.g. subcutaneously, intraperitoneally, by viralinfection, intravascularly, etc. Inhaled treatments are of particularinterest. Depending upon the manner of introduction, the compounds maybe formulated in a variety of ways. The concentration of therapeuticallyactive compound in the formulation may vary from about 0.1-100 wt. %.

The pharmaceutical compositions can be prepared in various forms, suchas granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing Agents, wetting and emulsifying Agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.

Pharmacogenetics

Pharmacogenetics is the linkage between an individual's genotype andthat individual's ability to metabolize or react to a therapeutic agent.Differences in metabolism or target sensitivity can lead to severetoxicity or therapeutic failure by altering the relation betweenbioactive dose and blood concentration of the drug. In the past fewyears, numerous studies have established good relationships betweenpolymorphisms in metabolic enzymes or drug targets, and both responseand toxicity. These relationships can be used to individualizetherapeutic dose administration.

Genotyping of polymorphic alleles is used to evaluate whether anindividual will respond well to a particular therapeutic regimen. Thepolymorphic sequences are also used in drug screening assays, todetermine the dose and specificity of a candidate therapeutic agent. Acandidate Nkx-2.2 polymorphism is screened with a target therapy todetermine whether there is an influence on the effectiveness intreating, for example, diabetes, depression, and/or obesity. Drugscreening assays are performed as described above. Typically two or moredifferent sequence polymorphisms are tested for response to a therapy.Therapies for diabetes currently include replacement therapy viaadministration of insulin and administration of drugs that increaseinsulin secretion (sulfonylureas) and drugs that reduce insulinresistance (such as troglitazone). Drugs currently used to treatdepression include serotonin uptake blockers (e.g, prozac), while drugscurrently used to treat obesity include fen-phen.

Where a particular sequence polymorphism correlates with differentialdrug effectiveness, diagnostic screening may be performed. Diagnosticmethods have been described in detail in a preceding section. Thepresence of a particular polymorphism is detected, and used to developan effective therapeutic strategy for the affected individual.

Detection of Nkx-2.2 Associated Disorders

Diagnosis of Nkx-2.2-associated disorders is performed by protein, DNAor RNA sequence and/or hybridization analysis of any convenient samplefrom a patient, e.g. biopsy material, blood sample, scrapings fromcheek, etc. A nucleic acid sample from a patient having a disorder thatmay be associated with Nkx-2.2, is analyzed for the presence of apredisposing polymorphism in Nkx-2.2. A typical patient genotype willhave at least one predisposing mutation on at least one chromosome. Thepresence of a polymorphic Nkx-2.2 sequence that affects the activity orexpression of the gene product, and confers an increased susceptibilityto an Nkx-2.2 associated disorder (e.g, hyperglycemia, diabetes,depression, or obesity) is considered a predisposing polymorphism.Individuals are screened by analyzing their DNA or mRNA for the presenceof a predisposing polymorphism, as compared to sequence from anunaffected individual(s). Specific sequences of interest include, forexample, any polymorphism that is associated with a diabetic syndrome,especially with Type 2 diabetes, or is otherwise associated withdiabetes, including, but not limited to, insertions, substitutions anddeletions in the coding region sequence, intron sequences that affectsplicing, or promoter or enhancer sequences that affect the activity andexpression of the protein.

Screening may also be based on the functional or antigeniccharacteristics of the protein. Immunoassays designed to detectpredisposing polymorphisms in Nkx-2.2 proteins may be used in screening.Where many diverse mutations lead to a particular disease phenotype,functional protein assays can be effective screening tools.

Biochemical studies may be performed to determine whether a candidatesequence polymorphism in the Nkx-2.2 coding region or control regions isassociated with disease. For example, a change in the promoter orenhancer sequence that affects expression of Nkx-2.2 may result inpredisposition to diabetes, depression, and/or obesity. Expressionlevels of a candidate variant allele are compared to expression levelsof the normal allele by various methods known in the art. Methods fordetermining promoter or enhancer strength include quantitation of theexpressed natural protein; insertion of the variant control element intoa vector with a reporter gene such as β-galactosidase, luciferase,chloramphenicol acetyltransferase, etc. that provides for convenientquantitation; and the like. The activity of the encoded Nkx-2.2 proteinmay be determined by comparison with the wild-type protein.

A number of methods are available for analyzing nucleic acids for thepresence of a specific sequence. Where large amounts of DNA areavailable, genomic DNA is used directly. Alternatively, the region ofinterest is cloned into a suitable vector and grown in sufficientquantity for analysis. Cells that express Nkx-2.2 genes, such aspancreatic cells, may be used as a source of mRNA, which may be assayeddirectly or reverse transcribed into cDNA for analysis. The nucleic acidmay be amplified by conventional techniques, such as the polymerasechain reaction (PCR), to provide sufficient amounts for analysis. Theuse of the polymerase chain reaction is described in Saiki, et al. 1985Science 239:487; a review of current techniques may be found inSambrook, et al. Molecular Cloning: A Laboratory Manual, CSH Press 1989,pp.14.2-14.33. Amplification may also be used to determine whether apolymorphism is present, by using a primer that is specific for thepolymorphism. Alternatively, various methods are known in the art thatutilize oligonucleotide ligation as a means of detecting polymorphisms,for examples see Riley et al. 1990 Nucl. Acid Res. 18:2887-2890; andDelahunty et al. 1996 Am. J. Hum. Genet. 58:1239-1246.

A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two stage system,where the amplified DNA is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g. avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

The sample nucleic acid, e.g. amplified or cloned fragment, is analyzedby one of a number of methods known in the art. The nucleic acid may besequenced by dideoxy or other methods, and the sequence of basescompared to either a neutral Nkx-2.2 sequence (e.g,. an Nkx-2.2 sequencefrom an unaffected individual). Hybridization with the variant sequencemay also be used to determine its presence, by Southern blots, dotblots, etc. The hybridization pattern of a control and variant sequenceto an array of oligonucleotide probes immobilized on a solid support, asdescribed in U.S. Pat. No. 5,445,934, or in WO95/35505, may also be usedas a means of detecting the presence of variant sequences. Single strandconformational polymorphism (SSCP) analysis, denaturing gradient gelelectrophoresis (DGGE), mismatch cleavage detection, and heteroduplexanalysis in gel matrices are used to detect conformational changescreated by DNA sequence variation as alterations in electrophoreticmobility. Alternatively, where a polymorphism creates or destroys arecognition site for a restriction endonuclease (restriction fragmentlength polymorphism, RFLP), the sample is digested with thatendonuclease, and the products size fractionated to determine whetherthe fragment was digested. Fractionation is performed by gel orcapillary electrophoresis, particularly acrylamide or agarose gels.

The hybridization pattern of a control and variant sequence to an arrayof oligonucleotide probes immobilized on a solid support, as describedin U.S. Pat. No. 5,445,934, or in WO95/35505, may be used as a means ofdetecting the presence of variant sequences. In one embodiment of theinvention, an array of oligonucleotides are provided, where discretepositions on the array are complementary to at least a portion of mRNAor genomic DNA of the Nkx-2.2 locus. Such an array may comprise a seriesof oligonucleotides, each of which can specifically hybridize to anucleic acid, e.g. mRNA, cDNA, genomic DNA, etc. from either the Nkx-2.2locus. Usually such an array will include at least 2 differentpolymorphic sequences, i.e. polymorphisms located at unique positionswithin the locus, usually at least about 5, more usually at least about10, and may include as many as 50 to 100 different polymorphisms. Theoligonucleotide sequence on the array will usually be at least about 12nt in length, may be the length of the provided polymorphic sequences,or may extend into the flanking regions to generate fragments of 100 to200 nt in length. For examples of arrays, see Hacia et al. 1996 NatureGenetics 14:441-447; Lockhart et al. 1996 Nature Biotechnol.14:1675-1680; and De Risi et al. 1996 Nature Genetics 14:457-460.

Antibodies specific for Nkx-2.2 polymorphisms may be used in screeningimmunoassays. A reduction or increase in Nkx-2.2 and/or presence of anNkx-2.2 disorder associated polymorphism is indicative that thesuspected disorder is Nkx-2.2-associated. A sample is taken from apatient suspected of having an Nkx-2.2-associated disorder. Samples, asused herein, include tissue biopsies, biological fluids, organ or tissueculture derived fluids, and fluids extracted from physiological tissues,as well as derivatives and fractions of such fluids. The number of cellsin a sample will generally be at least about 10³, usually at least 10⁴more usually at least about 10⁵. The cells may be dissociated, in thecase of solid tissues, or tissue sections may be analyzed. Alternativelya lysate of the cells may be prepared.

Diagnosis may be performed by a number of methods. The different methodsall determine the absence or presence or altered amounts of normal orabnormal Nkx-2.2 in patient cells suspected of having a predisposingpolymorphism in Nkx-2.2. For example, detection may utilize staining ofcells or histological sections, performed in accordance withconventional methods. The antibodies of interest are added to the cellsample, and incubated for a period of time sufficient to allow bindingto the epitope, usually at least about 10 minutes. The antibody may belabeled with radioisotopes, enzymes, fluorescers, chemiluminescers, orother labels for direct detection. Alternatively, a second stageantibody or reagent is used to amplify the signal. Such reagents arewell known in the art. For example, the primary antibody may beconjugated to biotin, with horseradish peroxidase-conjugated avidinadded as a second stage reagent. Final detection uses a substrate thatundergoes a color change in the presence of the peroxidase. The absenceor presence of antibody binding may be determined by various methods,including flow cytometry of dissociated cells, microscopy, radiography,scintillation counting, etc.

An alternative method for diagnosis depends on the in vitro detection ofbinding between antibodies and Nkx-2.2 in a lysate. Measuring theconcentration of Nkx-2.2 binding in a sample or fraction thereof may beaccomplished by a variety of specific assays. A conventional sandwichtype assay may be used. For example, a sandwich assay may first attachNkx-2.2-specific antibodies to an insoluble surface or support. Theparticular manner of binding is not crucial so long as it is compatiblewith the reagents and overall methods of the invention. They may bebound to the plates covalently or non-covalently, preferablynon-covalently.

The insoluble supports may be any compositions to which polypeptides canbe bound, which is readily separated from soluble material, and which isotherwise compatible with the overall method. The surface of suchsupports may be solid or porous and of any convenient shape. Examples ofsuitable insoluble supports to which the receptor is bound includebeads, e.g. magnetic beads, membranes and microtiter plates. These aretypically made of glass, plastic (e.g. polystyrene), polysaccharides,nylon or nitrocellulose. Microtiter plates are especially convenientbecause a large number of assays can be carried out simultaneously,using small amounts of reagents and samples.

Patient sample lysates are then added to separately assayable supports(for example, separate wells of a microtiter plate) containingantibodies. Preferably, a series of standards, containing knownconcentrations of normal and/or abnormal Nkx-2.2 is assayed in parallelwith the samples or aliquots thereof to serve as controls. Preferably,each sample and standard will be added to multiple wells so that meanvalues can be obtained for each. The incubation time should besufficient for binding, generally, from about 0.1 to 3 hr is sufficient.After incubation, the insoluble support is generally washed of non-boundcomponents. Generally, a dilute non-ionic detergent medium at anappropriate pH, generally 7-8, is used as a wash medium. From one to sixwashes may be employed, with sufficient volume to thoroughly washnon-specifically bound proteins present in the sample.

After washing, a solution containing a second antibody is applied. Theantibody will bind Nkx-2.2 with sufficient specificity such that it canbe distinguished from other components present. The second antibodiesmay be labeled to facilitate direct, or indirect quantification ofbinding. Examples of labels that permit direct measurement of secondreceptor binding include radiolabels, such as ³H or ¹²⁵I, fluorescers,dyes, beads, chemiluminescers, colloidal particles, and the like.Examples of labels which permit indirect measurement of binding includeenzymes where the substrate may provide for a colored or fluorescentproduct. In a preferred embodiment, the antibodies are labeled with acovalently bound enzyme capable of providing a detectable product signalafter addition of suitable substrate. Examples of suitable enzymes foruse in conjugates include horseradish peroxidase, alkaline phosphatase,malate dehydrogenase and the like. Where not commercially available,such antibody-enzyme conjugates are readily produced by techniques knownto those skilled in the art. The incubation time should be sufficientfor the labeled ligand to bind available molecules. Generally, fromabout 0.1 to 3 hr is sufficient, usually 1 hr sufficing.

After the second binding step, the insoluble support is again washedfree of non-specifically bound material. The signal produced by thebound conjugate is detected by conventional means. Where an enzymeconjugate is used, an appropriate enzyme substrate is provided so adetectable product is formed.

Other immunoassays are known in the art and may find use as diagnostics.Ouchterlony plates provide a simple determination of antibody binding.Western blots may be performed on protein gels or protein spots onfilters, using a detection system specific for Nkx-2.2 as desired,conveniently using a labeling method as described for the sandwichassay.

Other diagnostic assays of interest are based on the functionalproperties of Nkx-2.2 proteins. Such assays are particularly usefulwhere a large number of different sequence changes lead to a commonphenotype. For example, a functional assay may be based on thetranscriptional changes mediated by Nkx-2.2 gene products. Other assaysmay, for example, detect conformational changes, size changes resultingfrom insertions, deletions or truncations, or changes in the subcellularlocalization of Nkx-2.2 proteins.

In a protein truncation test, PCR fragments amplified from the Nkx-2.2gene or its transcript are used as templates for in vivotranscription/translation reactions to generate protein products.Separation by gel electrophoresis is performed to determine whether thepolymorphic gene encodes a truncated protein, where truncations may beassociated with a loss of function.

Diagnostic screening may also be performed for polymorphisms that aregenetically linked to a predisposition for diabetes, depression, and/orobesity, particularly through the use of microsatellite markers orsingle nucleotide polymorphisms. Frequently the microsatellitepolymorphism itself is not phenotypically expressed, but is linked tosequences that result in a disease predisposition. However, in somecases the microsatellite sequence itself may affect gene expression.Microsatellite linkage analysis may be performed alone, or incombination with direct detection of polymorphisms, as described above.The use of microsatellite markers for genotyping is well documented. Forexamples, see Mansfield et al. 1994 Genomics 24:225-233; Ziegle et al.1992 Genomics 14:1026-1031; Dib et al., supra.

Microsatellite loci that are useful in the subject methods have thegeneral formula:

U(R)_(n)U′,

where U and U′ are non-repetitive flanking sequences that uniquelyidentify the particular locus, R is a repeat motif, and n is the numberof repeats. The repeat motif is at least 2 nucleotides in length, up to7, usually 2-4 nucleotides in length. Repeats can be simple or complex.The flanking sequences U and U′ uniquely identify the microsatellitelocus within the human genome. U and U′ are at least about 18nucleotides in length, and may extend several hundred bases up to about1 kb on either side of the repeat. Within U and U′, sequences areselected for amplification primers. The exact composition of the primersequences are not critical to the invention, but they must hybridize tothe flanking sequences U and U′, respectively, under stringentconditions. Criteria for selection of amplification primers are aspreviously discussed. To maximize the resolution of size differences atthe locus, it is preferable to chose a primer sequence that is close tothe repeat sequence, such that the total amplification product isbetween 100-500 nucleotides in length.

The number of repeats at a specific locus, n, is polymorphic in apopulation, thereby generating individual differences in the length ofDNA that lies between the amplification primers. The number will varyfrom at least 1 repeat to as many as about 100 repeats or more.

The primers are used to amplify the region of genomic DNA that containsthe repeats. Conveniently, a detectable label will be included in theamplification reaction, as previously described. Multiplex amplificationmay be performed in which several sets of primers are combined in thesame reaction tube. This is particularly advantageous when limitedamounts of sample DNA are available for analysis. Conveniently, each ofthe sets of primers is labeled with a different fluorochrome.

After amplification, the products are size fractionated. Fractionationmay be performed by gel electrophoresis, particularly denaturingacrylamide or agarose gels. A convenient system uses denaturingpolyacrylamide gels in combination with an automated DNA sequencer, seeHunkapillar et al. 1991 Science 254:59-74. The automated sequencer isparticularly useful with multiplex amplification or pooled products ofseparate PCR reactions. Capillary electrophoresis may also be used forfractionation. A review of capillary electrophoresis may be found inLanders, et al. 1993 BioTechniques 14:98-111. The size of theamplification product is proportional to the number of repeats (n) thatare present at the locus specified by the primers. The size will bepolymorphic in the population, and is therefore an allelic marker forthat locus.

Therapeutic Uses of Nkx-2.2-Encoding Nucleic Acid

Nkx-2.2-encoding nucleic acid can be introduced into a cell toaccomplish transformation of the cell, preferably stable transformation,and the transformed cell subsequently implanted into a subject having adisorder characterized by a deficiency in insulin and/or serotoninproduction (e.g., an Nkx-2.2-associated disorder), depending upon thetissue into which the transformed cell is implanted. Preferably, thehost cell to be transformed and implanted in the subject is derived fromthe individual who will receive the transplant (e.g., to provide anautologous transplant). Where the transformed cells are to be insertedinto individual (e.g., into the pancreas, liver, abdominal cavity,etc.), the cells into which the nucleic acid is introduced arepreferably stem cells capable of developing into β cells within thepancreatic tissue environment, e.g., stem cells derived fromgastrointestinal tissue, or cells capable of expression of insulin uponexpression of the Nkx-2.2-encoding nucleic acid. Where the transformedcells are to be transplanted into the individual to provide increasedserotonin production, the cells into which the nucleic acid isintroduced are preferably a stem cell that is capable of developing intoa serotonin-secreting cell in a brain tissue environment, or a cellcapable of production of serotonin upon expression of Nkx-2.2-encodingnucleic acid.

For example, in a subject having Type 1 diabetes, gastrointestinal stemcells can be isolated from the affected subject, the cells transformedwith Nkx-2.2-encoding DNA, and the transformed cells implanted in theaffected subject to provide for insulin production. In a subjectsuffering from obesity and/or depression, a cell suitable forimplantation in brain tissue is transformed with Nkx-2.2-encoding DNA,transformed cells selected and expanded, and the transformed,Nkx-2.2-expressing cells implanted into the appropriate site in braintissue of the affected subject to provide for serotonin production.

Introduction of the Nkx-2.2-encoding nucleic acid into the cell can beaccomplished according to methods well known in the art (e.g., throughuse of electroporation, microinjection, lipofection infection with arecombinant (preferably replication-deficient) virus, and other meanswell known in the art). Preferably, the Nkx-2.2-encoding nucleic acid isoperably linked to a promoter that facilitates a desired level ofNkx-2.2 polypeptide expression (e.g., a promoter derived from CMV, SV40,adenovirus, or a tissue-specific or cell type-specific promoter).Transformed cells containing the Nkx-2.2-encoding nucleic acid can beselected and/or enriched via, for example, expression of a selectablemarker gene present in the Nkx-2.2-encoding construct or that is presenton a plasmid that is co-transfected with the Nkx-2.2-encoding construct.Typically selectable markers provide for resistance to antibiotics suchas tetracycline, hygromycin, neomycin, and the like. Other markers caninclude thymidine kinase and the like.

The ability of the transformed cells to express the Nkx-2.2-encodingnucleic acid can be assessed by various methods known in the art. Forexample, Nkx-2.2 expression can be examined by Northern blot to detectmRNA which hybridizes with a DNA probe derived from the relevant gene.Those cells that express the desired gene can be further isolated andexpanded in in vitro culture using methods well known in the art. Thehost cells selected for transformation with Nkx-2.2-encoding DNA willvary with the purpose of the ex vivo therapy (e.g., insulin productionor serotonin production), the site of implantation of the cells, andother factors that will vary with a variety of factors that will beappreciated by the ordinarily skilled artisan.

Methods for engineering a host cell for expression of a desired geneproduct(s) and implantation or transplantion of the engineered cells(e.g., ex vivo therapy) are known in the art (see, e.g., Gilbert et al.1993 “Cell transplantation of genetically altered cells on biodegradablepolymer scaffolds in syngeneic rats,” Transplantation 56:423-427). Forexample, for expression of a desired gene in exogenous or autologouscells and implantation of the cells for expression of the desired geneproduct in brain, see, e.g., Martinez-Serrano et al. 1995 “CNS-derivedneural progenitor cells for gene transfer of nerve growth factor to theadult rat brain: complete rescue of axotomized cholinergic neurons aftertransplantation into the septum,” J Neurosci 15:5668-5680; Taylor et al.1997 “Widespread engraftment of neural progenitor and stem-like cellsthroughout the mouse brain,” Transplant Proc 29:845-847; Snyder et al.1997 “Potential of neural “stem-like” cells for gene therapy and repairof the degenerating central nervous system,” Adv Neurol 1997;72:121-132;Snyder et al. 1996 “Gene therapy in neurology,” Curr Opin Pediatr 19968(6):558-568; Kordower et al. 1997 “Dopaminergic transplants in patientswith Parkinson's disease: neuroanatomical correlates of clinicalrecovery,” Exp Neurol 144:41-46; Lacorazza et al. 1996 “Expression ofhuman beta-hexosaminidase alpha-subunit gene (the gene defect ofTay-Sachs disease) in mouse brains upon engraftment of transducedprogenitor cells,” Nat Med 2:424-429; Martinez-Serrano et al. 1996 “Exvivo gene transfer of brain-derived neurotrophic factor to the intactrat forebrain: neurotrophic effects on cholinergic neurons,” Eur JNeurosci 8:727-735; Snyder 1995 “Immortalized neural stem cells:insights into development; prospects for gene therapy and repair,” ProcAssoc Am Physicians 107:195-204; Tuszynski et al. 1996 “Gene therapy inthe adult primate brain: intraparenchymal grafts of cells geneticallymodified to produce nerve growth factor prevent cholinergic neuronaldegeneration,” Gene Ther 3:305-314; and Karpati et al. 1996 “Theprinciples of gene therapy for the nervous system,” Trends Neurosci19:49-54.

For expression of a desired gene in exogenous or autologous cells andimplantation of the cells (e.g., islet cells) into pancreas, see, e.g.,Docherty 1997 “Gene therapy for diabetes mellitus,” Clin Sci (Colch)92:321-330; Hegre et al. 1976 “Transplantation of islet tissue in therat,” Acta Endocrinol Suppl (Copenh) 205:257-281; Sandler et al. 1997“Assessment of insulin secretion in vitro from microencapsulated fetalporcine islet-like cell clusters and rat, mouse, and human pancreaticislets,” Transplantation 63:1712-1718; Calafiore 1997 “Perspectives inpancreatic and islet cell transplantation for the therapy of IDDM,”Diabetes Care 20:889-896; Kenyon et al. 1996 “Islet celltransplantation: beyond the paradigms,” Diabetes Metab Rev 12:361-372;Sandler; Chick et al. 1977 Science “Artificial pancreas using livingbeta cells: effects on glucose homeostasis in diabetic rats,”197:780-782.

After expansion of the transformed cells in vitro, the cells areimplanted into the mammalian subject, preferably into the tissue fromwhich the cells were originally derived, by methods well known in theart. The number of cells implanted is a number of cells sufficient toprovide for expression of levels of Nkx-2.2 sufficient to provide forenhanced levels of insulin or serotonin production. The number cells tobe transplanted can be determined based upon such factors as the levelsof polypeptide expression achieved in vitro, and/or the number of cellsthat survive implantation. Preferably the cells are implanted in an areaof dense vascularization, and in a manner that minimizes evidence ofsurgery in the subject. The engraftment of the implant of transformedcells is monitored by examining the mammalian subject for classic signsof graft rejection, i.e., inflammation and/or exfoliation at the site ofimplantation, and fever.

Alternatively, Nkx-2.2-encoding nucleic acid can be delivered directlyto an affected subject to provide for Nkx-2.2 expression in a targetcell (e.g., a pancreatic cell, brain cell, gut cell, liver cell, orother organ cell capable of expressing Nkx-2.2 and providing productionor insulin or serotonin), thereby promoting development of the cell intoan insulin-producing cell (e.g., in pancreas) or a serotonin-producingcell (e.g., in brain) or to cure a defect in Nkx-2.2 expression in thesubject. Methods for in vivo delivery of a nucleic acid of interest forexpression in a target cell are known in the art. For example, in vivomethods of gene delivery normally employ either a biological means ofintroducing the DNA into the target cells (e.g., a virus containing theDNA of interest) or a mechanical means to introduce the DNA into thetarget cells (e.g., direct injection of DNA into the cells, liposomefusion, pneumatic injection using a “gene gun,” or introduction of theDNA via a duct of the pancreas). For other methods of introduction of aDNA of interest into a cell in vivo, also see Bartlett et al. 1997 “Useof biolistic particle accelerator to introduce genes into isolatedislets of Langerhans,” Transplant Proc 29:2201-2202; Furth 1997 “Genetransfer by biolistic process,” Mol Biotechnol 7:139-143; Gainer et al.1996 “Successful biolistic transformation of mouse pancreatic isletswhile preserving cellular function,” Transplantation 61:1567-1571;Docherty 1997 “Gene therapy for diabetes mellitus,” Clin Sci (Colch)92:321-330; Maeda et al. 1994 “Gastroenterology 1994“Adenovirus-mediated transfer of human lipase complementary DNA to thegallbladder,” 106:1638-1644.

The amount of DNA and/or the number of infectious viral particleseffective to infect the targeted tissue, transform a sufficient numberof cells, and provide for production of a desired level of insulin orserotonin can be readily determined based upon such factors as theefficiency of the transformation in vitro and the susceptibility of thetargeted secretory gland cells to transformation. For example, theamount of DNA injected into the pancreas of a human is, for example,generally from about 1 μg to 750 mg, preferably from about 500 μg to 500mg, more preferably from about 10 mg to 200 mg, most preferably about100 mg. Generally, the amounts of DNA can be extrapolated from theamounts of DNA effective for delivery and expression of the desired genein an animal model. For example, the amount of DNA for delivery in ahuman is roughly 100 times the amount of DNA effective in a rat.

Regardless of whether the Nkx-2.2-encoding DNA is introduced in vivo orex vivo, the DNA (or cells expressing the DNA) can be administered incombination with other genes and other agents. In addition,Nkx-2.2-encoding DNA (or recombinant cells expressing Nkx-2.2 DNA) canbe used therapeutically for disorders associated with, for example, adecrease in insulin production and/or serotonin production, but whichare not associated with an alteration in Nkx-2.2 function per se. Forexample, an increase in Nkx-2.2 may cause an increase in insulinproduction in the β cells of an individual that has decreased insulinproduction from some other cause not related to function of Nkx-2.2.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tocarry out the invention and is not intended to limit the scope of whatthe inventors regard as their invention. Efforts have been made toensure accuracy with respect to numbers used (e.g., amounts,temperatures, etc.), but some experimental error and deviation should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

Example 1

Isolation and Sequencing of a Human Nkx-2.2 Polypeptide-EncodingPolynucleotide

The GenBank non-redundant nucleotide sequence database(http://www.ncbi.nlm.nih.gov) was searched using the BLAST program forhuman sequences related to the mouse Nkx2.2 mRNA (GenBank Accession No.U31566). This analysis identified one human sequence, that of a CpGisland DNA genomic Mse 1 fragment, clone 60e5, (GenBank Accession No.Z65806) having 84% nucleotide sequence identity with mouse Nkx2.2 mRNAsequence in the region of the initiating methionine codon.

Two primers were prepared based on the sequence of clone 60e5 (Nkx2.2-P1and Nkx2.2-P2, (see Table 1 for sequences of all primers)) and used toisolate a P1-derived artifical chromosome (PAC) clone containing thehuman Nkx2.2 gene. PAC DNA pools (Genome Systems, St. Louis, Mo.) werescreened using the polymerase chain reaction (PCR) and primers Nkx2.2-P1and P2. The PCR conditions were initial denaturation at 94° C. for 5min, 35 cycles of denaturation at 94° C. for 30 sec, annealing at 65° C.for 30 sec and extension at 72° C. for 30 sec, and final extension at72° C. for 10 min. The PCR reaction contained 1 mM MgCl₂ and 5%dimethylsulfoxide in addition to the standard reagents. The PCR productswere separated by electrophoresis on a 4% NuSieve 3:1 agarose gel (FMCBioProducts, Rockland, Me.) and the 123 bp PCR product defined by theprimers Nkx2.2-P1 and P2 was visualized by staining the agarose gel withethidium bromide. One PAC clone, 310N11, was identified as containingthe human Nkx2.2 gene.

DNA was prepared from PAC 310N11 and digested with the restrictionendonucleases EcoRI, HindIII and Pst I. The restriction fragments wereseparated by electrophoresis on a 1% agarose gel and blotted to a Nylonmembrane which was hybridized with ³²P-labeled Nkx2.2-P1 and Nkx2.2-mP1(see Table 1); this sequence is complementary to mouse Nkx2.2 mRNA inthe region of codons 247-252). An approximately 8 kb HindIII fragmenthybridized to both primers suggesting that this fragment contained theentire human Nkx2.2 gene. The 8 kb HindIII fragment was subcloned intothe vector pBluescript II SK(+) and partially sequenced using primersNkx2.2-P1, P2, mP1, mP2 and primers based on newly determined humansequence (Table 1) as well as vector specific primers (T7 and T3). Thesequences were obtained using a AmpliTaq FS Dye Terminator CycleSequencing Kit (Perkin-Elmer, Norwalk, Conn.) and ABI PrismJ 377 DNASequencer (ABI, Foster City, Calif.). The partial sequence of the humanNkx2.2 gene determined in this manner is shown in FIGS. 1A and 1B. Ithas been deposited in the GenBank database with Accession Nos. AF019414and AF019415.

The chromosomal localization of the human Nkx2.2 was determined byfluorescence in situ hybridization to normal human metaphase chromsomesas described by Rowley et al 1990, Proc Natl Acad Sci USA 87:9358-9362using biotin-labeled PAC 310N11 DNA as a probe. Hybridization wasdetected with fluorescein-conjugated avidin (Vector Laboratories,Burlingame, Calif.) and chromosomes were identified by staining with4,6-diamidino-2-phenylindole-dihydrochloride (DAPI). Biotin-labeled PAC310N11 DNA specifically hybrized only to human chromosome 20. Specificlabeling of 20p11-p12 was observed on four (18 cells), three (6 cells)or two (1 cell) chromatids of the chromosome 20 homologues in 25 cellsexamined. Of 92 signals observed (92 of 100 20p chromatids werelabeled), all 92 (100%) were located at 20p11-p12. Of these, 84 (91%)were located at 20p11, 4 (4.5%) were located at the junction of20p11-p12, and 4 signals (4.5%) were located at 20p12. No backgroundsignals were observed at other chromosomal sites. These results localizethe human Nkx2.2 gene to chromosome 20, band p11.

The human Nkx2.2 gene consists of two exons which span a region of about3.5 kbp of human chromosome 20, band p11. The single intron interruptscodon 87. Exon 1 includes the 5′-untranslated region and codons 1-86 andthe first nucleotide of codon 87, and exon 2 includes the second andthird nucleotides of codon 87, codon 88-273 and the 3′-untranslatedregion. The amino acid sequence of Nkx2.2 is highly conserved and thereis 98% identity between the human and mouse orthologs. There is 93%nucleotide sequence identity between the protein coding regions of thehuman and mouse Nkx2.2 genes.

TABLE 1 Sequences of primers used for isolating and sequencing humanNkx2.2 gene Primer designation Sequence (5′-3′) Exon 1 Nkx2.2-P1ACGAATTGACCAAGTGAAGCTAC (SEQ ID NO:11) Nkx2.2-P2 CGTTGGTGTCCGGCAGGTCTAAG(SEQ ID NO:12) Nkx2.2-P5 AACCCGGGCTGCGGCTGCAGGAAT (SEQ ID NO:13) Exon 2Nkx2.2-mP1 AGCTGTACTGGGCGTTGTAC (SEQ ID NO:14) Nkx2.2-mP2TGACGAGTCACCGGACAATG (SEQ ID NO:15) Nkx2.2-P3 GCTCCAGCTCGTAGGTCTGCGCC(SEQ ID NO:16) Nkx2.2-P4 GCAGTCGCTGCAGCACATGC (SEQ ID NO:17) Nkx2.2-P6ATCCAGGGTGCTCCGAGTCTGGTGCA (SEQ ID NO:18) Nkx2.2-P7AGTCGAGTTGACTCTCGGCTCCAC (SEQ ID NO:19) Nkx2.2-P9GTGGAGCCGAGAGTCAACTCGACT (SEQ ID NO:20) Nkx2.2-P10ACGCAGGTCAAGATCTGGTTCCAG (SEQ ID NO:21)

The genomic sequence of human Nkx-2.2 is shown in FIGS. 1A and 1B. Thehuman Nkx-2.2 sequence is composed of two exons (SEQ ID NOS:3 and 4)separated by a single intron (illustrated in FIGS. 1A and 1B as twosequences (SEQ ID NOS: 8 and 9) separated by approximately 1.2 kb ofadditional intronic sequence). When spliced, exon 1 (SEQ ID NO:3) andexon 2 (SEQ ID NO:4) are joined to form the coding sequence SEQ ID NO:1,which encodes the full-length human Nkx-2.2 polypeptide (SEQ ID NO:2).The last nucleotide of exon 1 and the first two nucleotides of exon twoare joined to form a codon encoding a leucine at residue position 87 inthe Nkx-2.2 amino acid sequence (SEQ ID NO:2). In addition to the exonand intronic sequences, the sequenced clone included approximately 104nucleotides of 5′ untranslated sequence and about 851 nucleotides of 3′untranslated sequence.

Example 2

Heterozygous Nkx-2.2 Knock-Out Transgenic Mice

To determine the function of Nkx-2.2 in development, mice heterozygousfor an Nkx-2.2 mutation, as well as mice homozygous for a null mutationof Nkx-2.2 (see Example 2 below) were generated using standard genetargeting techniques. Briefly, an Nkx-2.2 gene targeting vector wasconstructed by isolating several genomic DNA clones containing theentire Nkx-2.2 gene from a 129J mouse genomic library using PCR primersbased on the rat homeobox and NK-2 box (Price et al. 1992 Neuron8:241-255). Two partially overlapping genomic clones were isolated; eachclone contained an approximately 14 kb insert containing the Nkx-2.2gene in a relatively central position within each clone. These genomicclones were mapped extensively with restriction enzymes and Southernanalysis. This mapping data provided sufficient information for theconstruction of an Nkx-2.2 gene replacement vector.

A null mutation of Nkx-2.2 was generated by replacing all of the codingsequence of Nkx-2.2, including the homeobox region, with a PGK-neocassette. Approximately 6 kb of 3′ and 4 kb of 5′ Nkx-2.2 genomicsequences flanked the deletion in order to facilitate homologousrecombination events. The recombinant plasmid also contained a herpessimplex virus thymidine kinase (tk) gene flanking the genomic DNA topermit selection against nonhomologous recombination events.

The Nkx-2.2 targeting DNA vector was electroporated into mouse (129J)embryonic stem (ES) cells using the positive-negative selection strategyfor homologous recombination (Mansour, et al. 1988 Nature 336:348-352).Candidate ES clones were screened for homologous recombination bySouthern analysis of the genomic DNA. Four clones contained the mutantallele and had undergone a single homologous integration at the Nkx-2.2locus.

Correctly targeted ES cells were injected into C57BL/6J host blastocyststo generate chimeric mice. Chimeric males that transmit the targeted DNAthrough the germline will be bred to produce Fl mice that areheterozygous for each of the mutant alleles. Inbreeding between theheterozygous mice will be used to produce homozygous mutant animals.

Heterozygous Nkx-2.2 knock-out transgenics survived to adulthood, werefertile, and have an apparently normal phenotype in all assays tested.The Nkx-2.2 heterozygous knock-outs may have decreased insulin andinsulin mRNA levels compared to wildtype mice. Obese heterozygotes havebeen observed at 1 year after birth.

Example 3

Homozygous Nkx-2.2 Knock-Out Transgenic Mice

Homozygous Nkx-2.2 knock-out transgenic mice were produced by crossingthe heterozygous transgenic mice described in Example 1.

Homozygous knock-out (null) Nkx-2.2. mice were grossly indistinguishablefrom their wildtype and heterozygous littermates at birth. However, bythe third day after birth, the homozygous mutant animals displayedgrowth retardation and did not survive longer than six days postnatally.The gross morphology of the homozygous null Nkx-2.2 transgenic miceappeared normal. However, histological analysis revealed a generalreduction in islet cell mass; the exocrine cells appeared unaffected.Immunohistochemical analysis showed that there was a remarkable defectin islet cell development. Insulin was undetectable in comparativestudies between mutant and wildtype littermates. In addition, the numberof cells producing glucagon were reduced, and the level of glucagon percell was diminished. Glucokinase expression in the pancreas wasundetectable. Radioimmunoassays confirmed the immunohistochemicalresults. Insulin content in the pancreas of Nkx-2.2 null mice wasundetectable, and glucagon content was reduced at least 20-fold relativeto wildtype mice.

A large population of cells within clusters of the pancreas did notproduce any of the four endocrine hormones (insulin, glucagon,somatostatin, and pancreatic polypeptide). Because expression ofpancreatic polypeptide and somatostatin in the pancreas as a whole wasnormal, these non-endocrine producing cells, which appeared withinnormal size islets, may be precursors to the glucagon-producing a orinsulin-producing β cells. The null Nkx-2.2 animals still exhibitedroughly normal islet amyloid polypeptide (amylin) expression. A summaryof the molecular characteristics of islets in the Nkx-2.2 nulltransgenic animals (as determined by immunohistochemistry) is providedin Table 2 below.

TABLE 2 Islet Molecular Characteristics in the Nkx-2.2 Mutant-Summary ofImmunohistochemistry Results Endocrine Hormones Insulin undetectableGlucagon reduced Somatostatin normal Pancreatic polypeptide reduced βCell Markers Glucokinase undetectable Glut2 undetectable PC1/PC3 normalAmylin normal Transcription Factors Pax6 normal/reduced Pdx1 reducedNkx-6.1 (putative transcription factor) undetectable

In addition to the defects in the pancreas, the homozygous Nkx-2.2knock-out transgenic mice also exhibited neural defects. A subset ofserotonin-producing cells in the brain were decreased in number relativeto the number of serotonin-producing cells in wildtype mice. These datashow that Nkx-2.2 plays a critical role in islet development, insulinand glucagon synthesis, and in development of serotonin-producing cellsin the brain.

Example 3

Heterozygous Nkx-6.1 Knock-Out Transgenic Mice

Heterozygous Nkx-6.1 knock-out mice were generated as described inExample 1 except that the gene targeting vector containedNkx-6.1-encoding DNA rather than Nkx-2.2-encoding DNA. Briefly, anNkx-6.1 gene targeting vector was constructed by isolating a genomic DNAclones containing the Nkx-6.1 gene from a 129J mouse genomic libraryusing the hamster Nkx-6.1 cDNA sequence as a probe (Rudnick et al. 1994Proc. Natl. Acad. Sci. USA 91:12203-12207). A single clone was obtainedthat included the entire coding region of the Nkx-6.1 gene. Whenintroduced into ES cells as described above, the targeting vectorreplaced the first exon (which contained the translation start site)with a neomycin resistance gene. Rather than using the tk- selectiondescribed in Example 1, ES colonies were simply screened by Southernblot analysis for homologous integration events. The ES cells were thenimplanted into blastocysts, chimeras identified, and heterozygotesproduced as described above.

Heterozygous Nkx-6.1 knock-out transgenics survived to adulthood, werefertile, and have an apparently normal phenotype in all assays tested.

Example 4

Homozygous Nkx-6.1 Knock-Out Transgenic Mice

Homozygous Nkx-6.1 knock-out transgenic mice were produced by crossingthe heterozygous Nkx-6.1 knock-out transgenic mice described in Example3.

The pancreas of the homozygous knock-out (null) Nkx-6.1 transgenic micehad almost no β-cells, but other islet cells appeared normal. The nullNkx-6.1 mice died immediately after birth (within about 1 hour) andexhibited severe neurological defects, including decreased movement anddefects in motor neurons.

These data show that Nkx-6.1 plays a critical role in islet development,insulin production, and neural development.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

21 822 base pairs nucleic acid single linear unknown Coding Sequence1...819 1 ATG TCG CTG ACC AAC ACA AAG ACG GGG TTT TCG GTC AAG GAC ATCTTA 48 Met Ser Leu Thr Asn Thr Lys Thr Gly Phe Ser Val Lys Asp Ile Leu 15 10 15 GAC CTG CCG GAC ACC AAC GAT GAG GAG GGC TCT GTG GCC GAA GGT CCG96 Asp Leu Pro Asp Thr Asn Asp Glu Glu Gly Ser Val Ala Glu Gly Pro 20 2530 GAG GAA GAG AAC GAG GGG CCC GAG CCA GCC AAG AGG GCC GGG CCG CTG 144Glu Glu Glu Asn Glu Gly Pro Glu Pro Ala Lys Arg Ala Gly Pro Leu 35 40 45GGG CAG GGC GCC CTG GAC GCG GTG CAG AGC CTG CCC CTG AAG AAC CCC 192 GlyGln Gly Ala Leu Asp Ala Val Gln Ser Leu Pro Leu Lys Asn Pro 50 55 60 TTCTAC GAC AGC AGC GAC AAC CCG TAC ACG CGC TGG CTG GCC AGC ACC 240 Phe TyrAsp Ser Ser Asp Asn Pro Tyr Thr Arg Trp Leu Ala Ser Thr 65 70 75 80 GAGGGC CTT CAG TAC TCC CTG CAC GGT CTG GCT GCC GGG GCG CCC CCT 288 Glu GlyLeu Gln Tyr Ser Leu His Gly Leu Ala Ala Gly Ala Pro Pro 85 90 95 CAG GACTCA AGC TCC AAG TCC CCG GAG CCC TCG GCC GAC GAG TCA CCG 336 Gln Asp SerSer Ser Lys Ser Pro Glu Pro Ser Ala Asp Glu Ser Pro 100 105 110 GAC AATGAC AAG GAG ACC CCG GGC GGC GGG GGG GAC GCC GGC AAG AAG 384 Asp Asn AspLys Glu Thr Pro Gly Gly Gly Gly Asp Ala Gly Lys Lys 115 120 125 CGA AAGCGG CGA GTG CTT TTC TCC AAG GCG CAG ACC TAC GAG CTG GAG 432 Arg Lys ArgArg Val Leu Phe Ser Lys Ala Gln Thr Tyr Glu Leu Glu 130 135 140 CGG CGCTTT CGG CAG CAG CGG TAC CTG TCG GCG CCC GAG CGC GAA CAC 480 Arg Arg PheArg Gln Gln Arg Tyr Leu Ser Ala Pro Glu Arg Glu His 145 150 155 160 CTGGCC AGC CTC ATC CGC CTC ACG CCC ACG CAG GTC AAG ATC TGG TTC 528 Leu AlaSer Leu Ile Arg Leu Thr Pro Thr Gln Val Lys Ile Trp Phe 165 170 175 CAGAAC CAC CGC TAC AAG ATG AAG CGC GCC CGG GCC GAG AAA GGT ATG 576 Gln AsnHis Arg Tyr Lys Met Lys Arg Ala Arg Ala Glu Lys Gly Met 180 185 190 GAGGTG ACG CCC CTG CCC TCG CCG CGC CGG GTG GCC GTG CCC GTC TTG 624 Glu ValThr Pro Leu Pro Ser Pro Arg Arg Val Ala Val Pro Val Leu 195 200 205 GTCAGG GAC GGC AAA CCA TGT CAC GCG CTC AAA GCC CAG GAC CTG GCA 672 Val ArgAsp Gly Lys Pro Cys His Ala Leu Lys Ala Gln Asp Leu Ala 210 215 220 GCCGCC ACC TTC CAG GCG GGC ATT CCC TTT TCT GCC TAC AGC GCG CAG 720 Ala AlaThr Phe Gln Ala Gly Ile Pro Phe Ser Ala Tyr Ser Ala Gln 225 230 235 240TCG CTG CAG CAC ATG CAG TAC AAC GCC CAG TAC AGC TCG GCC AGC ACC 768 SerLeu Gln His Met Gln Tyr Asn Ala Gln Tyr Ser Ser Ala Ser Thr 245 250 255CCC CAG TAC CCG ACA GCA CAC CCC CTG GTC CAG GCC CAG CAG TGG ACT 816 ProGln Tyr Pro Thr Ala His Pro Leu Val Gln Ala Gln Gln Trp Thr 260 265 270TGG TGA 822 Trp 273 amino acids amino acid single linear proteininternal unknown 2 Met Ser Leu Thr Asn Thr Lys Thr Gly Phe Ser Val LysAsp Ile Leu 1 5 10 15 Asp Leu Pro Asp Thr Asn Asp Glu Glu Gly Ser ValAla Glu Gly Pro 20 25 30 Glu Glu Glu Asn Glu Gly Pro Glu Pro Ala Lys ArgAla Gly Pro Leu 35 40 45 Gly Gln Gly Ala Leu Asp Ala Val Gln Ser Leu ProLeu Lys Asn Pro 50 55 60 Phe Tyr Asp Ser Ser Asp Asn Pro Tyr Thr Arg TrpLeu Ala Ser Thr 65 70 75 80 Glu Gly Leu Gln Tyr Ser Leu His Gly Leu AlaAla Gly Ala Pro Pro 85 90 95 Gln Asp Ser Ser Ser Lys Ser Pro Glu Pro SerAla Asp Glu Ser Pro 100 105 110 Asp Asn Asp Lys Glu Thr Pro Gly Gly GlyGly Asp Ala Gly Lys Lys 115 120 125 Arg Lys Arg Arg Val Leu Phe Ser LysAla Gln Thr Tyr Glu Leu Glu 130 135 140 Arg Arg Phe Arg Gln Gln Arg TyrLeu Ser Ala Pro Glu Arg Glu His 145 150 155 160 Leu Ala Ser Leu Ile ArgLeu Thr Pro Thr Gln Val Lys Ile Trp Phe 165 170 175 Gln Asn His Arg TyrLys Met Lys Arg Ala Arg Ala Glu Lys Gly Met 180 185 190 Glu Val Thr ProLeu Pro Ser Pro Arg Arg Val Ala Val Pro Val Leu 195 200 205 Val Arg AspGly Lys Pro Cys His Ala Leu Lys Ala Gln Asp Leu Ala 210 215 220 Ala AlaThr Phe Gln Ala Gly Ile Pro Phe Ser Ala Tyr Ser Ala Gln 225 230 235 240Ser Leu Gln His Met Gln Tyr Asn Ala Gln Tyr Ser Ser Ala Ser Thr 245 250255 Pro Gln Tyr Pro Thr Ala His Pro Leu Val Gln Ala Gln Gln Trp Thr 260265 270 Trp 259 base pairs nucleic acid single linear unknown CodingSequence 1...258 3 ATG TCG CTG ACC AAC ACA AAG ACG GGG TTT TCG GTC AAGGAC ATC TTA 48 Met Ser Leu Thr Asn Thr Lys Thr Gly Phe Ser Val Lys AspIle Leu 1 5 10 15 GAC CTG CCG GAC ACC AAC GAT GAG GAG GGC TCT GTG GCCGAA GGT CCG 96 Asp Leu Pro Asp Thr Asn Asp Glu Glu Gly Ser Val Ala GluGly Pro 20 25 30 GAG GAA GAG AAC GAG GGG CCC GAG CCA GCC AAG AGG GCC GGGCCG CTG 144 Glu Glu Glu Asn Glu Gly Pro Glu Pro Ala Lys Arg Ala Gly ProLeu 35 40 45 GGG CAG GGC GCC CTG GAC GCG GTG CAG AGC CTG CCC CTG AAG AACCCC 192 Gly Gln Gly Ala Leu Asp Ala Val Gln Ser Leu Pro Leu Lys Asn Pro50 55 60 TTC TAC GAC AGC AGC GAC AAC CCG TAC ACG CGC TGG CTG GCC AGC ACC240 Phe Tyr Asp Ser Ser Asp Asn Pro Tyr Thr Arg Trp Leu Ala Ser Thr 6570 75 80 GAG GGC CTT CAG TAC TCC C 259 Glu Gly Leu Gln Tyr Ser 85 86amino acids amino acid single linear protein internal unknown 4 Met SerLeu Thr Asn Thr Lys Thr Gly Phe Ser Val Lys Asp Ile Leu 1 5 10 15 AspLeu Pro Asp Thr Asn Asp Glu Glu Gly Ser Val Ala Glu Gly Pro 20 25 30 GluGlu Glu Asn Glu Gly Pro Glu Pro Ala Lys Arg Ala Gly Pro Leu 35 40 45 GlyGln Gly Ala Leu Asp Ala Val Gln Ser Leu Pro Leu Lys Asn Pro 50 55 60 PheTyr Asp Ser Ser Asp Asn Pro Tyr Thr Arg Trp Leu Ala Ser Thr 65 70 75 80Glu Gly Leu Gln Tyr Ser 85 563 base pairs nucleic acid single linearunknown Coding Sequence 3...560 5 TG CAC GGT CTG GCT GCC GGG GCG CCC CCTCAG GAC TCA AGC TCC AAG 47 His Gly Leu Ala Ala Gly Ala Pro Pro Gln AspSer Ser Ser Lys 1 5 10 15 TCC CCG GAG CCC TCG GCC GAC GAG TCA CCG GACAAT GAC AAG GAG ACC 95 Ser Pro Glu Pro Ser Ala Asp Glu Ser Pro Asp AsnAsp Lys Glu Thr 20 25 30 CCG GGC GGC GGG GGG GAC GCC GGC AAG AAG CGA AAGCGG CGA GTG CTT 143 Pro Gly Gly Gly Gly Asp Ala Gly Lys Lys Arg Lys ArgArg Val Leu 35 40 45 TTC TCC AAG GCG CAG ACC TAC GAG CTG GAG CGG CGC TTTCGG CAG CAG 191 Phe Ser Lys Ala Gln Thr Tyr Glu Leu Glu Arg Arg Phe ArgGln Gln 50 55 60 CGG TAC CTG TCG GCG CCC GAG CGC GAA CAC CTG GCC AGC CTCATC CGC 239 Arg Tyr Leu Ser Ala Pro Glu Arg Glu His Leu Ala Ser Leu IleArg 65 70 75 CTC ACG CCC ACG CAG GTC AAG ATC TGG TTC CAG AAC CAC CGC TACAAG 287 Leu Thr Pro Thr Gln Val Lys Ile Trp Phe Gln Asn His Arg Tyr Lys80 85 90 95 ATG AAG CGC GCC CGG GCC GAG AAA GGT ATG GAG GTG ACG CCC CTGCCC 335 Met Lys Arg Ala Arg Ala Glu Lys Gly Met Glu Val Thr Pro Leu Pro100 105 110 TCG CCG CGC CGG GTG GCC GTG CCC GTC TTG GTC AGG GAC GGC AAACCA 383 Ser Pro Arg Arg Val Ala Val Pro Val Leu Val Arg Asp Gly Lys Pro115 120 125 TGT CAC GCG CTC AAA GCC CAG GAC CTG GCA GCC GCC ACC TTC CAGGCG 431 Cys His Ala Leu Lys Ala Gln Asp Leu Ala Ala Ala Thr Phe Gln Ala130 135 140 GGC ATT CCC TTT TCT GCC TAC AGC GCG CAG TCG CTG CAG CAC ATGCAG 479 Gly Ile Pro Phe Ser Ala Tyr Ser Ala Gln Ser Leu Gln His Met Gln145 150 155 TAC AAC GCC CAG TAC AGC TCG GCC AGC ACC CCC CAG TAC CCG ACAGCA 527 Tyr Asn Ala Gln Tyr Ser Ser Ala Ser Thr Pro Gln Tyr Pro Thr Ala160 165 170 175 CAC CCC CTG GTC CAG GCC CAG CAG TGG ACT TGG TGA 563 HisPro Leu Val Gln Ala Gln Gln Trp Thr Trp 180 185 186 amino acids aminoacid single linear protein internal unknown 6 His Gly Leu Ala Ala GlyAla Pro Pro Gln Asp Ser Ser Ser Lys Ser 1 5 10 15 Pro Glu Pro Ser AlaAsp Glu Ser Pro Asp Asn Asp Lys Glu Thr Pro 20 25 30 Gly Gly Gly Gly AspAla Gly Lys Lys Arg Lys Arg Arg Val Leu Phe 35 40 45 Ser Lys Ala Gln ThrTyr Glu Leu Glu Arg Arg Phe Arg Gln Gln Arg 50 55 60 Tyr Leu Ser Ala ProGlu Arg Glu His Leu Ala Ser Leu Ile Arg Leu 65 70 75 80 Thr Pro Thr GlnVal Lys Ile Trp Phe Gln Asn His Arg Tyr Lys Met 85 90 95 Lys Arg Ala ArgAla Glu Lys Gly Met Glu Val Thr Pro Leu Pro Ser 100 105 110 Pro Arg ArgVal Ala Val Pro Val Leu Val Arg Asp Gly Lys Pro Cys 115 120 125 His AlaLeu Lys Ala Gln Asp Leu Ala Ala Ala Thr Phe Gln Ala Gly 130 135 140 IlePro Phe Ser Ala Tyr Ser Ala Gln Ser Leu Gln His Met Gln Tyr 145 150 155160 Asn Ala Gln Tyr Ser Ser Ala Ser Thr Pro Gln Tyr Pro Thr Ala His 165170 175 Pro Leu Val Gln Ala Gln Gln Trp Thr Trp 180 185 104 base pairsnucleic acid single linear unknown 7 TCCCTCCCCC ACTCCCCCCT CCCCCGCCCGCCGGGGCAGG GGAGCGCCAC GAATTGACCA 60 AGTGAAGCTA CAACTTTGCG ACATAAATTTTGGGGTCTCG AACC 104 212 base pairs nucleic acid single linear unknown 8GTAAGTAGCA AAACTTGGCT GCCGAGGCCG TGGTCCCCTC CATTCCTGCA GCCGCAGCCC 60GGGTTGGACG CTGGGAGTGA AAGGGGAAGG GGCCATGTAA GCCCGGACCC CCTCACTCGG 120ATCCGTAGAA AGATTTTTAA CACCTGTATA GGATGTCCTC TGCCCTCCTC TTCAAGCCTC 180CTTAGTTCCG GGAAAGAACT TGGTCTCCAA AA 212 345 base pairs nucleic acidsingle linear unknown 9 TGCCCACGTC TCGCCGGAGA GGAAACCGCT TAAGGGCGCCGGAGCCCTTA ACCCGCGATG 60 ATCTTCAGTG CCACTTCCCC CCCCAAATTC TCACCCACACTATGTGAGCC CCTTGAAAGG 120 CGAGCCCCTA GCCCCCACTC CTACGGATTT CCCTCTTTACCCTGGGAGGT CCCGACGTCT 180 TCGTCAGGCG TAGAGGAAGG CAGGGGTCAT GGCAAAGGCAGCGGGGCTGG GCTGCCAGGC 240 GCGGAGGTCC AGGGTCGCAC GGAGGATCCA GGGTGCTCCGAGTCTGGTGC AGGCTGCGCG 300 CGGCCTCCAG ACGCCTGACG CGCTTCTCTC TCCCCCTCCCCCCAG 345 851 base pairs nucleic acid single linear unknown 10GCGCCGCCCC AACGAGACTC GCGGCCCCAG GCCCAGGCCC CACCCCGGCG GCGGTGGCGG 60CGAGGAGGCC TCGGTCCTTA TGGTGGTTAT TATTATTATT ATAATTATTA TTATGGAGTC 120GAGTTGACTC TCGGCTCCAC TAGGGAGGCG CCGGGAGGTT GCCTGCGTCT CCTTGGAGTG 180GCAGATTCCA CCCACCCAGC TCTGCCCATG CCTCTCCTTC TGAACCTTGG GAGAGGGCTG 240AACTCTACGC CGTGTTTACA GAATGTTTGC GCAGCTTCGC TTCTTTGCCT CTCCCCGGGG 300GGACCAAACC GTCCCAGCGT TAATGTCGTC ACTTGAAAAC GAGAAAAAGA CCGACCCCCC 360ACCCCTGCTT TCGTGCATTT TGTAAAATAT GTTTGTGTGA GTAGCGATAT TGTCAGCCGT 420CTTCTAAAGC AAGTGGAGAA CACTTTAAAA ATACAGAGAA TTTCTTCCTT TTTTTAAAAA 480AAAATAAGAA AATGCTAAAT ATTTATGGCC ATGTAAACGT TCTGACAACT GGTGGCAGAT 540TTCGCTTTTC GTTGTAAATA TCGGTGGTGA TTGTTGCCAA AATGACCTTC AGGACCGGCC 600TGTTTCCCGT CTGGGTCCAA CTCCTTTCTT TGTGGCTTGT TTGGGTTTGT TTTTTGTTTT 660GTTTTTGTTT TTGCGTTTTC CCCTGCTTTC TTCCTTTCTC TTTTTATTTT ATTGTGCAAA 720CATTTCTCAA ATATGGAAAA GAAAACCCTG TAGGCAGGGA GCCCTCTGCC CTGTCCTCCG 780GGCCTTCAGC CCCGAACTTG GAGCTCAGCT ATTCGGCGCG GTTCCCCAAC AGCGCCGGGC 840GCAGAAAGCT T 851 23 base pairs nucleic acid single linear unknown 11ACGAATTGAC CAAGTGAAGC TAC 23 23 base pairs nucleic acid single linearunknown 12 CGTTGGTGTC CGGCAGGTCT AAG 23 24 base pairs nucleic acidsingle linear unknown 13 AACCCGGGCT GCGGCTGCAG GAAT 24 20 base pairsnucleic acid single linear unknown 14 AGCTGTACTG GGCGTTGTAC 20 20 basepairs nucleic acid single linear unknown 15 TGACGAGTCA CCGGACAATG 20 23base pairs nucleic acid single linear unknown 16 GCTCCAGCTC GTAGGTCTGCGCC 23 20 base pairs nucleic acid single linear unknown 17 GCAGTCGCTGCAGCACATGC 20 26 base pairs nucleic acid single linear unknown 18ATCCAGGGTG CTCCGAGTCT GGTGCA 26 24 base pairs nucleic acid single linearunknown 19 AGTCGAGTTG ACTCTCGGCT CCAC 24 24 base pairs nucleic acidsingle linear unknown 20 GTGGAGCCGA GAGTCAACTC GACT 24 24 base pairsnucleic acid single linear unknown 21 ACGCAGGTCA AGATCTGGTT CCAG 24

What is claimed is:
 1. An isolated polynucleotide sequence, orcomplement thereof, comprising a polynucleotide sequence encoding ahuman Nkx-2.2 polypeptide.
 2. An isolated polynucleotide sequence, orcomplement thereof, comprising a polynucleotide sequence encoding ahuman Nkx-2.2 exon 1 polypeptide.
 3. An isolated polynucleotidesequence, or complement thereof, comprising a polynucleotide sequenceencoding a human Nkx-2.2 exon 2 polypeptide.
 4. An isolatedpolynucleotide sequence of claim 1 comprising a polynucleotide sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQID NO:5.
 5. An isolated polynucleotide sequence of claim 2 comprisingSEQ ID NO:3 or a degenerate variant of SEQ ID NO:3.
 6. An isolatedpolynucleotide sequence of claim 3 comprising SEQ ID NO:5 or adegenerate variant of SEQ ID NO:5.
 7. An isolated polynucleotide, orcomplement thereof, comprising SEQ ID NO:7.
 8. An isolatedpolynucleotide or complement thereof comprising a polynucleotidesequence encoding an amino acid sequence selected from the groupconsisting of: SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6.
 9. An isolatedpolynucleotide comprising a sequence of at least 200 contiguousnucleotides of a polynucleotide sequence selected from the groupconsisting of: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:10.10. An isolated polynucleotide or complement thereof comprising apolynucleotide sequence comprising at least 200 contiguous nucleotidesof a polynucleotide sequence encoding an amino acid sequence selectedfrom the group consisting of: SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6.11. A recombinant expression vector comprising the polynucleotidesequence of claim
 1. 12. An isolated recombinant host cell containingthe polynucleotide sequence of claim
 1. 13. A method for producing thehuman Nkx-2.2 polypeptide of claim 1, the method comprising the stepsof: a) culturing a recombinant host cell containing a human Nkx-2.2polypeptide-encoding polynucleotide sequence under conditions suitablefor the expression of the polypeptide; and b) recovering the polypeptidefrom the host cell culture.